Immunoglobulin constant region Fc receptor binding agents

ABSTRACT

IVIG replacement compounds are derived from recombinant and/or biochemical creation of immunologically active biomimetic(s). These replacement compounds are then screened in vitro to assess each replacements compound&#39;s efficiency at modulating immune function. Particular replacement compounds are selected for further in vivo validation and dosage/administration optimization. Finally, the replacement compounds are used to treat a wide range of diseases, including inflammatory and autoimmune diseases.

This application is a continuation of U.S. application Ser. No.12/602,609 filed Jun. 4, 2010, now U.S. Pat. No. 8,680,237, issued onMar. 25, 2014, which is a National Stage tiling of PCT/US2008/065428under 35 U.S.C. §371 which Claims priority to U.S. Provisional Appl. No.61/015,547, filed Dec. 20, 2007, U.S. Provisional Appl. No. 61/015,127,tiled Dec. 19, 2007 and U.S. Provisional Appl. No. 60/941,044, filedJun. 1, 2007, each of which are incorporated herein in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the fields of immunology,inflammation, and tumor immunology. More specifically, the presentinvention relates to biologically active biomimetic molecules comprisingimmunoglobulin Fc domains, compositions comprising such biomimetics, andmethods of using such biomimetics.

The invention also relates to the treatment and prophylaxis ofpathological conditions mediated by monocyte-derived cells, and moreparticularly to the use of stabilized functional portions of IgG Fcfragments for such treatment and prophylaxis.

2. Description of the Background Art

Immune globulin products from human plasma have been used since theearly 1950's to treat immune deficiency disorders and more recently, andmore commonly, for autoimmune and inflammatory diseases.

Initially, immune globulin products were administered by intramuscularinjection. More recently, intravenous immune globulin (IVIG) has beenused and was initially shown to be effective in treatment of theautoimmune disease idiopathic thrombocytopenic purpura (ITP) (Imbach P,Barandun S, d'Apuzzo V, et al: High-dose intravenous gammaglobulin foridiopathic thrombocytopenic purpura in childhood. Lancet 1981 Jun. 6;1(8232): 1228-31). Human IVIG (referred to herein as “hIVIG”) is aformulation of sterile, purified immunoglobulin G (IgG) productsmanufactured from pooled human plasma that typically contains more than95% unmodified IgG, with only small and variable amounts ofimmunoglobulin A (IgA) or immunoglobulin M (IgM) (see, for example,Rutter A, Luger T A: High-dose intravenous immunoglobulins: an approachto treat severe immune-mediated and autoimmune diseases of the skin. JAm Acad Dermatol 2001 June; 44(6): 1010-24). Today the single mostcommon clinical use of hIVIG is in the treatment of ITP.

While hIVIG has been an effective clinical treatment, there are severalshortcomings to hIVIG formulations, including the potential forinadequate sterility, the presence of impurities, lack of availability,and lot-to-lot variation. In particular hIVIG preparations can varygreatly in their immunoglobulin A (IgA) content which can be of concernbecause IgA can cause allergic and anaphylactic reactions inIgA-deficient recipients.

In view of the negative aspects of hIVIG, there exists a need for animproved means of treating autoimmune and inflammatory diseases.

In addition, multiple pathological conditions of a wide variety of typesare mediated by cells derived from monocytes. A simple therapeuticand/or prophylactic agent for use in many, if not all, such conditionswould be invaluable.

SUMMARY OF THE INVENTION

The immuno-regulatory properties of WIG reside in the Fc domain of IgGmolecules. For example, in murine models of ITP, both unmodified IVIGand the Fc fragment alone demonstrate therapeutic efficacy in restoringplatelet counts, while isolated IVIG Fab fragments are not therapeutic(Samuelsson, A., Towers, T. L. & Ravetch, J. V. Anti-inflammatoryActivity of IVIG Mediated Through the Inhibitory Fc Receptor. Science291, 484-486 (2001)). Moreover Fc, but not Fab fragments of IVIG, isalso therapeutically effective in the treatment of both childhood andadult idiopathic thrombocytopenic purpura (Follea, G. et al. Intravenousplasmin-treated gammaglobulin therapy in idiopathic thrombocytopenicpurpura. Nouv Rev Fr Hematol 27, 5-10 (1985); Solal-Celigny, P.,Bernard, J., Herrera, A. & Biovin, P. Treatment of adult autoimmunethrombocytopenic purpura with high-dose intravenous plasmin-cleavedgammaglobulins. Scand J Haematol 31, 39-44 (1983); Debre, M. & Bonnet,M.-C. Infusion of Gcgamma fragments for treatment of children with acuteimmune thrombocytopenic purpura. Lancet 342, 945-49 (1993); Burdach, S.E., Evers, K. & Geurson, R. Treatment of acute idiopathicthrombocytopenic purpura of childhood with intravenous immunoglobulin G:Comparative efficacy of 7S and 5S preparations. J Pediatr 109, 770-775(1986)).

The therapeutic effect of IVIG is initially mediated through the Fcgamma receptor (FcγR) and relies on Dendritic Cell (DC)-macrophagecross-talk for its long term tolerogenic effects. FcγRIIIa plays arequisite role in the initiator phase and FcγRIIb is required for theeffector phase in murine models of ITP (Samuelsson, A., Towers, T. L. &Ravetch, J. V. Anti-inflammatory Activity of IVIG Mediated Through theInhibitory Fc Receptor. Science 291, 484-486 (2001); Siragam, V. et al.Intravenous immunoglobulin ameliorates ITP via activating Fc[gamma]receptors on dendritic cells. Nat Med 12, 688 (2006)). Similarly, humanstudies demonstrate that anti-Fcγ receptor antibodies are effective inthe treatment of refractory ITP (Clarkson, S. et al. Treatment ofrefractory immune thrombocytopenic purpura with an anti-Fcgamma-receptor antibody. N Engl J Med 314, 1236-1239 (1986)).Importantly, long term tolerogenic effects are mediated by cell-cellinteractions, as adoptive transfer of IVIG-treated DCs is effective intreating murine models of ITP (Siragam, V. et al. Intravenousimmunoglobulin ameliorates ITP via activating Fc[gamma] receptors ondendritic cells. Nat Med 12, 688 (2006)).

The immunomodulatory effects of IVIG require aggregation of the FcγR.Aggregation of FcγR is mediated by IgG dimers present in IVIG (5-15% ofthe total IVIG) (Bleeker, W. K. et al. Vasoactive side effects ofintravenous immunoglobulin preparations in a rat model and theirtreatment with recombinant platelet-activating factor acetylhydrolase.Blood 95, 1856-1861 (2000)). For example, in a murine model of ITP,treatment with IVIG with a high content of “dimers” (dimers of wholeimmunoglobulin molecules) enhanced platelet counts while IVIG “monomers”(whole immunoglobulin molecules) were not effective (Teeling, J. L. etal. Therapeutic efficacy of intravenous immunoglobulin preparationsdepends on the immunoglobulin G dimers: studies in experimental immunethrombocytopenia. Blood 98, 1095-1099 (2001)). Furthermore, despite thefact that ion exchange resin and polyethylene glycol fractionation areroutinely used in the manufacture of IVIG to remove IgG aggregates, theclinical efficacy of IVIG correlates with the presence of dimers in thepatient's sera (Augener, W., Friedman, B. & Brittinger, G. Areaggregates of IgG the effective part of high-dose immunoglobulin therapyin adult idiopathic thrombocytopenic purpura (ITP)? Blut 50, 249-252(1985)). Importantly, the perccntagc of dimers also correlates withvasoactive side effects, which are treatable with acetylhydrolase(Bleeker, W. K. et al. Vasoactive side effects of intravenousimmunoglobulin preparations in a rat model and their treatment withrecombinant platelet-activating factor acetylhydrolase. Blood 95,1856-1861 (2000)).

The present invention relates to biologically active biomimeticmolecules, compositions comprising the same, and methods of using thesame. These biomimetics have broad application for treatingimmunological and inflammatory disorders including but not limited toautoimmune diseases, and they have utility as bioimmunotherapy agentsfor cancer. Further, certain of these biomimetics also have utility asreagents, such as for use in immunological assays for testing immunecell function and in the diagnosis of disease. Moreover, the biomimeticsand compositions of the present invention have the advantage ofovercoming the above-listed limitations of hIVIG. The invention alsorelates to the treatment and prophylaxis of pathological conditionsmediated by monocyte-derived cells, and more particularly to the use ofstabilized functional portions of IgG Fc fragments for such treatmentand prophylaxis.

In a first embodiment the present invention is directed to isolatedserial stradomers comprising two or more associated stradomer monomers,wherein each of the stradomer monomers comprises two or more Fc domainmonomers, wherein the association of the two or more stradomer monomersforms two or more Fc domains, and wherein the serial stradomerspecifically binds to a first Fcγ receptor through a first of the two ormore Fc domains and to a second Fcγ receptor through a second of the twoor more Fc domains. In a preferred embodiment, the two or more stradomermonomers are associated through a covalent bond, a disulfide bond orchemical cross-linking.

In a preferred embodiment of the isolated serial stradomers of thepresent invention, the isolated serial stradomers are comprised of twoassociated stradomer monomers. In an equally preferred embodiment, theisolated serial stradomers are comprised of two associated stradomermonomers wherein both of the stradomer monomers comprise two Fc domainmonomers, and wherein the association of the two stradomer monomersforms two Fc domains. In a first particular example of these embodimentsdirected to isolated serial stradomers, at least one of the two Fcdomains comprises an IgG hinge and an IgG CH2 domain. In a secondparticular example each of the two Fc domains independently comprises anIgG hinge and an IgG CH2 domain. In a third particular example at leastone of the two Fc domains comprises an IgG hinge, an IgG CH2 domain andan IgG CH3 domain. In a fourth particular example each of the two Fcdomains independently comprises an IgG hinge, an IgG CH2 domain and anIgG CH3 domain. In a fifth particular example at least one of the two Fcdomains comprises an IgG1 hinge or an IgG3 hinge, an IgG1 CH2 domain oran IgG3 CH2 domain, and an IgG1 CH3 domain or an IgG3 CH3 domain. In asixth particular example at least one of the two Fc domains comprises anIgG1 hinge or an IgG3 hinge, and an IgG1 CH2 domain or an IgG3 CH2domain. In a seventh particular example each of the two Fc domainsindependently comprises an IgG1 hinge or an IgG3 hinge, an IgG1 CH2domain or an IgG3 CH2 domain, and an IgG1 CH3 domain or an IgG3 CH3domain. In an eighth particular example each of the two Fc domainsindependently comprises an IgG1 hinge, an IgG1 CH2 domain, and an IgG1CH3 domain. In a ninth particular example each of the two Fc domainsindependently comprises an IgG3 hinge, an IgG3 CH2 domain, and an IgG3CH3 domain. In a tenth particular example each of the two Fc domainsindependently comprises an IgG1 hinge, an IgG1 CH2 domain, and an IgG3CH3 domain.

Also in this first embodiment, the two or more Fc domains are each of asame immunoglobulin Fc class, and the immunoglobulin Fc class isselected from the group consisting of IgG1, IgG2, IgG3, and IgG4.Alternatively, the two or more Fc domains are each of a differentimmunoglobulin Fc class, and said immunoglobulin Fc class is selectedfrom the group consisting of IgG1, IgG2, IgG3 and IgG4.

Further in this first embodiment, the first and second Fcγ receptors areeach independently an Fcγ receptor I, an Fcγ receptor II, an Fcγreceptor III or an Fcγ receptor IV. Preferably the first and second Fcγreceptors are each Fcγ receptor IIIa.

In a second embodiment the present invention is directed to isolatedserial stradomers comprising two associated stradomer monomers, whereineach of the stradomer monomers comprises two Fc domain monomers, whereinthe association of the two stradomer monomers forms two Fc domains,wherein each of said two Fc domains independently comprises an IgGhinge, an IgG CH2 domain and an IgG CH3 domain, and wherein the serialstradomer specifically binds to a first Fcγ receptor through a first ofthe two Fc domains and to a second Fcγ receptor through a second of thetwo Fc domains. In a preferred embodiment, the two or more stradomermonomers are associated through a covalent bond, a disulfide bond orchemical cross-linking.

In a first particular example of this second embodiment the two Fcdomains are each of a same immunoglobulin Fc class, and theimmunoglobulin Fc class is selected from the group consisting of IgG1,IgG2, IgG3, and IgG4. In a second particular example the two Fc domainsare each of a different immunoglobulin Fc class, and said immunoglobulinFc class is selected from the group consisting of IgG1, IgG2, IgG3 andIgG4. In a third particular example at least one of the Fc domainscomprises an IgG hinge and an IgG CH2 domain. In a fourth particularexample each of the Fc domains independently comprises an IgG hinge andan IgG CH2 domain. In a fifth particular example at least one of the Fcdomains comprises an IgG hinge, an IgG CH2 domain and an IgG CH3 domain.In a sixth particular example each of the Fc domains independentlycomprises an IgG hinge, an IgG CH2 domain and an IgG CH3 domain. In aseventh particular example at least one of the Fc domains comprises anIgG1 hinge or an IgG3 hinge, an IgG1 CH2 domain or an IgG3 CH2 domain,and an IgG1 CH3 domain or an IgG3 CH3 domain. In an eighth particularexample each of the Fc domains independently comprises an IgG1 hinge oran IgG3 hinge, an IgG1 CH2 domain or an IgG3 CH2 domain, and an IgG1 CH3domain or an IgG3 CH3 domain. In a ninth particular example each of theFc domains independently comprises an IgG1 hinge, an IgG1 CH2 domain,and an IgG1 CH3 domain. In a tenth particular example each of the Fcdomains independently comprises an IgG3 hinge, an IgG3 CH2 domain, andan IgG3 CH3 domain. In an eleventh particular example each of the Fcdomains independently comprises an IgG1 hinge, an IgG1 CH2 domain, andan IgG3 CH3 domain.

In a third embodiment the present invention is directed to isolatedserial stradomers further comprising a Fab domain, wherein each of thestradomer monomers comprises an Fab fragment heavy chain and two Fcdomain monomers, wherein the Fab fragment heavy chain is in a positionamino terminal or carboxy terminal to the two Fc domain monomers,wherein an Fab fragment light chain is independently associated witheach Fab fragment heavy chain, and wherein the Fab domain hasantigen-binding activity. In a preferred embodiment, each of thestradomer monomers further comprises an immunoglobulin hinge monomer,and wherein the immunoglobulin hinge monomer is in a position betweenthe Fab fragment heavy chain and the two Fc domain monomers.

In a fourth embodiment the present invention is directed to corestradomers comprising a core moiety linked to two or more core stradomerunits, wherein each of the two or more core stradomer units comprises atleast one Fc domain, and wherein each of the core stradomer units isindependently selected from the group consisting of:

(a) an Fc fragment, wherein said Fc fragment comprises two associated Fcfragment monomers, wherein each of said Fc fragment monomers comprisesan Fc domain monomer, and wherein the association of the two Fc fragmentmonomers forms an Fc domain,

(b) an Fc partial fragment, wherein said Fc partial fragment comprisestwo associated Fc partial fragment monomers, wherein each of said Fcpartial fragment monomers comprises an Fc domain monomer, and whereinthe association of the two Fc partial fragment monomers forms an Fcdomain,

(c) an Fc domain, wherein said Fc domain comprises two associated Fcdomain monomers, and wherein the association of the two Fc domainmonomers forms an Fc domain,

(d) a serial stradomer, wherein said serial stradomer comprises two ormore associated stradomer monomers, wherein each of said stradomermonomers comprises two or more Fc domain monomers, and wherein theassociation of the two or more stradomer monomers forms two or more Fcdomains, and

(e) a cluster stradomer, wherein said cluster stradomer comprises two ormore multimerized cluster stradomer units, wherein each of said clusterstradomer units comprises a multimerizing region and at least one Fcdomain, wherein each of said cluster stradomer units comprises twoassociated cluster stradomer unit monomers, wherein each of said clusterstradomer unit monomers comprises a multimerizing region monomer and atleast one Fc domain monomer, wherein the association of the two clusterstradomer unit monomers forms a multimerizing region and at least one Fcdomain, and wherein the multimerizing regions of the two or more clusterstradomer units multimerize to form the cluster stradomer, and

wherein the core stradomer specifically binds to a first Fcγ receptorthrough a first of the two or more core stradomer units and to a secondFcγ receptor through a second of the two or more core stradomer units.

Preferably in this fourth embodiment, the core moiety is selected fromthe group consisting of an immunoglobulin J chain, albumin, liposome,bead, peptide and polyethylene glycol.

In preferred embodiments directed to core stradomers the two or morecore stradomer units are each independently an Fc fragment.Alternatively, the two or more core stradomer units are eachindependently a serial stradomer.

In a further preferred embodiment directed to core stradomers the corestradomer comprises two core stradomer units, wherein each of the twocore stradomer units is each independently a serial stradomer, whereinthe serial stradomer comprises two associated stradomer monomers,wherein both of said stradomer monomers comprises two Fc domainmonomers, and wherein the association of the two stradomer monomersforms two Fc domains. In a first particular example of this embodiment,at least one of the Fc domains of the two or more core stradomer unitscomprises an IgG1 hinge or an IgG3 hinge, an IgG1 CH2 domain or an IgG3CH2 domain, and an IgG1 CH3 domain or an IgG3 CH3 domain. In a secondparticular example at least one of the Fc domains of the two or more twocore stradomer units comprises an IgG1 hinge or an IgG3 hinge, and anIgG1 CH2 domain. In a third particular example each of the Fc domains ofthe two or more two core stradomer units independently comprises an IgG1hinge, an IgG1 CH2 domain, and an IgG1 CH3 domain. In a fourthparticular example at least one of the Fc domains of the two or more twocore stradomer units comprises an IgG hinge and an IgG CH2 domain. In afifth particular example each of the Fc domains of the two or more twocore stradomer units independently comprises an IgG hinge and an IgG CH2domain. In a sixth particular example each of the Fc domains of the twoor more two core stradomer units independently comprises an IgG3 hinge,an IgG3 CH2 domain, and an IgG3 CH3 domain. In a seventh particularexample each of the Fc domains of the two or more two core stradomerunits independently comprises an IgG1 hinge, an IgG1 CH2 domain, and anIgG3 CH3 domain.

In this embodiment, the first and second Fcγ receptors are eachindependently an Fcγ receptor I, an Fcγ receptor II, an Fcγ receptor IIIor an Fcγ receptor IV. Preferably, the first and second Fcγ receptorsare each Fcγ receptor IIIa.

In a fifth embodiment the present invention is directed to clusterstradomers comprising two or more multimerized cluster stradomer units,wherein each of the cluster stradomer units comprises a multimerizingregion and at least one Fc domain, wherein each of the cluster stradomerunits comprises two associated cluster stradomer unit monomers, whereineach of the cluster stradomer unit monomers comprises a multimerizingregion monomer and at least one Fc domain monomer, wherein theassociation of the two cluster stradomer unit monomers forms amultimerizing region and at least one Fc domain, wherein themultimerizing regions of the two or more cluster stradomer unitsmultimerize to form the cluster stradomer, and wherein the clusterstradomer specifically binds to a first Fcγ receptor through a first Fcdomain and to a second Fcγ receptor through a second Fc domain.

In preferred embodiments, the multimerizing region is selected from thegroup consisting of an IgG2 hinge, an IgE CH2 domain, a leucine, anisoleucine zipper and a zinc finger.

In further preferred embodiment, the cluster stradomers comprising two,three, four or five multimerized cluster stradomer units.

In a first particular example of this fifth embodiment at least one ofthe Fc domains comprises an IgG1 hinge or an IgG3 hinge, an IgG1 CH2domain or an IgG3 CH2 domain, and an IgG1 CH3 domain or an IgG3 CH3domain. In a second particular example each of the Fc domainsindependently comprises an IgG1 hinge, an IgG1 CH2 domain, and an IgG1CH3 domain. In a third particular example at least one of the Fc domainscomprises an IgG hinge and an IgG CH2 domain. In a fourth particularexample each of the Fc domains independently comprises an IgG hinge andan IgG CH2 domain. In a fifth particular example each of the Fc domainsindependently comprises an IgG3 hinge, an IgG3 CH2 domain, and an IgG3CH3 domain. In a sixth particular example each of the Fc domainsindependently comprises an IgG1 hinge, an IgG1 CH2 domain, and an IgG3CH3 domain. In a seventh particular example each of the Fc domainsindependently comprises an IgG hinge, an IgG CH2 domain and an IgG CH3domain. In an eighth particular example at least one of the clusterstradomer units comprises two or more Fc domains. In a ninth particularexample each of the cluster stradomer units comprises two or more Fcdomains.

In this embodiment, the first and second Fcγ receptors are eachindependently an Fcγ receptor I, an Fcγ receptor II, an Fcγ receptor IIIor an Fcγ receptor IV. Preferably, the first and second Fcγ receptorsare each Fcγ receptor IIIa.

In a sixth embodiment the present invention is directed to stradobodiescomprising two or more associated stradomer monomers and an Fab domain,wherein each of the stradomer monomers comprises an Fab fragment heavychain and two or more Fc domain monomers, wherein the Fab fragment heavychain is in a position amino terminal or carboxy terminal to the two ormore Fc domain monomers, wherein the association of the two or morestradomer monomers forms two or more Fc domains, wherein an Fab fragmentlight chain is independently associated with the Fab fragment heavychain of each stradomer monomer, wherein the Fab domain hasantigen-binding activity, and wherein the stradobody specifically bindsto a first Fcγ receptor through a first of the two or more Fc domainsand to a second Fcγ receptor through a second of the two or more Fcdomains.

In preferred embodiments the two or more stradomer monomers areassociated through a covalent bond, a disulfide bond or chemicalcross-linking.

In a further preferred embodiment, each of said stradomer monomers ofthe stradobodies further comprises an immunoglobulin hinge monomer, andwherein the immunoglobulin hinge monomer is in a position between theFab fragment heavy chain and the two Fc domain monomers.

In a particular embodiment the stradobody comprises two associatedstradomer monomers, wherein each of said stradomer monomers comprises anFab fragment heavy chain and two Fc domain monomers, and wherein theassociation of the two stradomer monomers forms two Fc domains. In afirst particular example of this embodiment, at least one of the two Fcdomains comprises an IgG hinge, an IgG CH2 domain and an IgG CH3 domain.In a second particular example each of the two Fc domains independentlycomprises an IgG hinge, an IgG CH2 domain and an IgG CH3 domain. In athird particular example at least one of the two Fc domains comprises anIgG hinge and an IgG CH3 domain. In a fourth particular example each ofthe Fc two domains independently comprises an IgG hinge and an IgG CH3domain. In a fifth particular example at least one of the two Fc domainscomprises an IgG1 hinge or an IgG3 hinge, an IgG1 CH2 domain or an IgG3CH2 domain, and an IgG1 CH3 domain or an IgG3 CH3 domain. In a sixthparticular example each of the two Fc domains independently comprises anIgG1 hinge or an IgG3 hinge, an IgG1 CH2 domain or an IgG3 CH2 domain,and an IgG1 CH3 domain or an IgG3 CH3 domain. In a seventh particularexample each of the two Fc domains independently comprises an IgG1hinge, an IgG1 CH2 domain, and an IgG1 CH3 domain. In an eighthparticular example each of the two Fc domains independently comprises anIgG3 hinge, an IgG3 CH2 domain, and an IgG3 CH3 domain. In a ninthparticular example each of the two Fc domains independently comprises anIgG1 hinge, an IgG1 CH2 domain, and an IgG3 CH3 domain. In a tenthparticular example at least one of the two Fc domains comprises an IgG1hinge or an IgG3 hinge, and an IgG1 CH2 domain or an IgG3 CH2 domain. Inan eleventh particular example at least one of the two Fc domainscomprises an IgG1 hinge or an IgG3 hinge, and an IgG1 CH2 domain.

In this embodiment, the first and second Fcγ receptors are eachindependently an Fcγ receptor I, an Fcγ receptor II, an Fcγ receptor IIIor an Fcγ receptor IV. Preferably, the first and second Fcγ receptorsare each Fcγ receptor IIIa.

In a seventh embodiment the present invention is directed to methods ofaltering an immune response in a subject comprising administering to asubject in need thereof a pharmaceutical composition comprising atherapeutically effective amount of a serial stradomer and a carrier ordiluent. In a preferred embodiment, the pharmaceutical compositioncomprises a therapeutically effective amount of a heterogeneous mixtureof serial stradomers and a carrier or diluent.

In an eighth embodiment the present invention is directed to methods ofaltering an immune response in a subject comprising administering to asubject in need thereof a pharmaceutical composition comprising atherapeutically effective amount of a core stradomer and a carrier ordiluent. In a preferred embodiment, the pharmaceutical compositioncomprises a therapeutically effective amount of a heterogeneous mixtureof core stradomers and a carrier or diluent.

In a ninth embodiment the present invention is directed to methods ofaltering an immune response in a subject comprising administering to asubject in need thereof a pharmaceutical composition comprising atherapeutically effective amount of a cluster stradomer and a carrier ordiluent. In a preferred embodiment, the pharmaceutical compositioncomprises a therapeutically effective amount of a heterogeneous mixtureof cluster stradomers and a carrier or diluent.

In a tenth embodiment the present invention is directed to methods ofaltering an immune response in a subject comprising administering to asubject in need thereof a pharmaceutical composition comprising atherapeutically effective amount of a stradobody and a carrier ordiluent. In a preferred embodiment, the pharmaceutical compositioncomprises a therapeutically effective amount of a heterogeneous mixtureof stradobodies and a carrier or diluent.

In an eleventh embodiment the present invention is directed to methodsof screening an antibody for a specific activity on a cell of the immunesystem, comprising: (a) contacting a homogenous population of cells ofthe immune system with a candidate antibody, (b) measuring an activityof the population of cells of (a), (c) contacting a homogenouspopulation of cells of the same cell type as in (a) with a serialstradomer of claim 1, (d) measuring an activity of the population ofcells of (c), and (e) comparing the activity measured in (b) with theactivity measured in (d), thereby screening an antibody for a specificactivity on a cell of the immune system. In a preferred embodiment, thecandidate antibody and the serial stradomer are species-matched andisotype-matched. In a further preferred embodiment, the comparison in(e) is a ratio of activity measured in (d) versus the activity measuredin (b).

In a twelfth embodiment the present invention is directed methods ofinhibiting the activity of a monocyte-derived cell (MDC). The methodinvolves contacting the cell with a composition containing a substratewith an Fc reagent bound to it. The contacting can be in vitro, in vivo,or ex vivo. The cell can be in an animal, e.g., an animal that has or isat risk of developing a monocyte derived cell mediated condition(MDCMC). The cell can be, for example, a dendritic cell, a macrophage, amonocyte, or an osteoclast.

In a thirteenth embodiment the present invention is directed methods oftreatment that includes administering to an animal a compositioncomprising a substrate having an Fc reagent bound thereto, the animalhaving or being at risk of developing a monocyte-derived cell mediatedcondition (MDCMC).

The following are embodiments common to both these two methods (thetwelfth and thirteenth embodiments).

The animal can be, for example, a human.

The Fc reagent can contain or be a functional portion of a human Fcfragment, e.g., a human IgG1 Fc fragment, a human IgG3 Fc fragment, ahuman IgG2, or a human IgG4 Fc fragment. Moreover it can include or bean IgG molecule. The Fc reagent can also be or include a functionalportion of a non-human Fc fragment.

The substrate can be or include a synthetic polymer, e.g., nylon,teflon, dacron, polyvinyl chloride, PEU (poly(ester urethane)), PTFE(polytetrafluoroethylene), or PMMA (methyl methacrylate). The substratecan include or be a metal or a metal alloy, e.g., stainless steel,platinum, iridium, titanium, tantalum, a nickel-titanium alloy, or acobalt-chromium alloy. The substrate can contain or be animal tissue oran animal tissue product, e.g., a tissue or organ graft, bone (e.g.,osteogenic bone), or cartilage. The substrate can contain or be aprotein, e.g., collagen or keratin. The substrate can also be or containa polysaccharide, e.g., agarose. Moreover, the substrate can contain orbe a tissue matrix, e.g., an acellular tissue matrix. The substrate cancontain or be an animal cell (e.g., a tissue repair cell such as afibroblasts or a mesenchymal stem cell). The substrate can contain or bea salt, e.g., calcium sulfate. Furthermore the substrate can be orcontain a gel or cream. It can also contain or be silicon or silastic.It can also contain be a natural fiber, e.g., silk, cotton, or wool.

The substrate can be a hair transplant plug or an implantable medicaldevice such as a stent (e.g., a vascular stent such as a coronary arterystent; an airway stent such as an endotracheal or nasal stent; agastrointestinal stent such a biliary or pancreatic stent; or a urinarystent such as a ureteral stent). It can also be a surgical suture (e.g.,a braid silk, chromic gut, nylon, plastic, or metal suture or a surgicalclip (e.g., an aneurism clip)). In addition, the substrate the can be anartificial hip, an artificial hip joint, an artificial knee, anartificial knee joint, an artificial shoulder, an artificial shoulderjoint, an artificial finger or toe joint, a bone plate, a bone dowel, abone non-union implant, an intervertebral disk implant, bone cement, ora bone cement spacer. It can be an arterial-venous shunt, an implantablewire, a pacemaker, an artificial heart, a heart assist device, acochlear implant, an implantable defibrillator, a spinal cordstimulator, a central nervous system stimulator, a peripheral nerveimplant, a dental prosthesis, or a dental crown. Furthermore, thesubstrate can be a large vessel embolic filtering device or cage, apercutaneous device, a dermal or sub-mucosal patch, or an implantabledrug delivery device.

The substrate can also be a large blood vessel graft, wherein the bloodvessel is, for example, a carotid artery, a femoral artery, or an aorta.It can also be a sub-dermal implant, a corneal implant, an intraocularlens, or a contact lens.

The substrate can be in the form of, e.g., a sheet, a bead, a mesh, apowder particle, a thread, a bead, or a fiber. The substrate can containor be a solid, a semi-solid, or a gelatinous substance. Thus, asubstrate includes substances that are substantially insoluble inaqueous solvents, e.g., a fat-soluble lipid such as a liposome.

The MDCMC can be an inflammatory condition, an autoimmune disease, acancer, a disorder of bone density, an acute infection, or a chronicinfection.

It can be a hematoimmunological process, e.g., IdiopathicThrombocytopenic Purpura, alloimmune/autoimmune thrombocytopenia,Acquired immune thrombocytopenia, Autoimmune neutropenia, Autoimmunehemolytic anemia, Parvovirus B19-associated red cell aplasia, Acquiredantifactor VIII autoimmunity, acquired von Willebrand disease, MultipleMyeloma and Monoclonal Gammopathy of Unknown Significance, Sepsis,Aplastic anemia, pure red cell aplasia, Diamond-Blackfan anemia,hemolytic disease of the newborn, Immune-mediated neutropenia,refractoriness to platelet transfusion, neonatal post-transfusionpurpura, hemolytic uremic syndrome, systemic Vasculitis, Thromboticthrombocytopenic purpura, or Evan's syndrome.

Alternatively, the MDCMC can be a neuroimmunological process, e.g.,Guillain-Barré syndrome, Chronic Inflammatory DemyelinatingPolyradiculoneuropathy, Paraproteinemic IgM demyelinatingPolyneuropathy, Lambert-Eaton myasthenic syndrome, Myasthenia gravis,Multifocal Motor Neuropathy, Lower Motor Neuron Syndrome associated withanti-GM1 antibodies, Demyelination, Multiple Sclerosis and opticneuritis, Stiff Man Syndrome, Paraneoplastic cerebellar degenerationwith anti-Yo antibodies, paraneoplastic encephalomyelitis, sensoryneuropathy with anti-Hu antibodies, epilepsy, Encephalitis, Myelitis,Myelopathy especially associated with Human T-cell lymphotropic virus-1,Autoimmune Diabetic Neuropathy, or Acute Idiopathic DysautonomicNeuropathy.

The MDCMC can be a Rheumatic disease process, e.g., Kawasaki's disease,Rheumatoid arthritis, Felty's syndrome, ANCA-positive Vasculitis,Spontaneous Polymyositis, Dermatomyositis, Antiphospholipid syndromes,Recurrent spontaneous abortions, Systemic Lupus Erythematosus, Juvenileidiopathic arthritis, Raynaud's, CREST syndrome, or Uveitis.

Moreover, the MDCMC can be a dermatoimmunological disease process, e.g.,Epidermal Necrolysis, Gangrene, Granuloma, Autoimmune skin blisteringdiseases including Pemphigus vulgaris, Bullous Pemphigoid, and Pemphigusfoliaccus, Vitiligo, Streptococcal toxic shock syndrome, Scleroderma,systemic sclerosis including diffuse and limited cutaneous systemicsclerosis, Atopic dermatitis, or steroid dependent Atopic dermatitis.

In addition, the MDCMC can be a musculoskeletal immunological disease,e.g., Inclusion Body Myositis, Necrotizing fasciitis, InflammatoryMyopathies, Myositis, Anti-Decorin (BJ antigen) Myopathy, ParaneoplasticNecrotic Myopathy, X-linked Vacuolated Myopathy, Penacillamine-inducedPolymyositis, Atherosclerosis, Coronary Artery Disease, orCardiomyopathy.

The MDCMC can also be a gastrointestinal immunological disease process,e.g., pernicious anemia, autoimmune chronic active hepatitis, primarybiliary cirrhosis, Celiac disease, dermatitis herpetiformis, cryptogeniccirrhosis, Reactive arthritis, Crohn's disease, Whipple's disease,ulcerative colitis, or sclerosing cholangitis.

The MDCMC can be, for example, Graft Versus Host Disease,Antibody-mediated rejection of the graft, Post-bone marrow transplantrejection, Post-infectious disease inflammation, Lymphoma, Leukemia,Neoplasia, Asthma, Type 1 Diabetes mellitus with anti-beta cellantibodies, Sjogren's syndrome, Mixed Connective Tissue Disease,Addison's disease, Vogt-Koyanagi-Harada Syndrome, Membranoproliferativeglomerulonephritis, Goodpasture's syndrome, Graves' disease, Hashimoto'sthyroiditis, Wegener's granulomatosis, micropolyarterits, Churg-Strausssyndrome, Polyarteritis nodosa or Multisystem organ failure.

Where the MDCMC is a cancer, it can be fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom'smacroglobulinemia, myelodysplastic disease, heavy chain disease,neuroendocrine tumors, or Schwanoma.

Where the MDCMC is a disorder of bone density, it can be osteoporosis,osteopenia, osteopetrosis, idiopathic hypogonadotropic hypogonadism,anorexia nervosa, non-healing fracture, post-menopausal osteoporosis,Vitamin D deficiency or excess, primary or secondaryhyperparathyroidism, thyroid disease, or bisphosphonate toxicity.

Where the MDCMC is an acute infection, it can be: a fungal disorderincluding Candidiasis, Candidemia, or Aspergillosis; a bacterialdisorder, including staphylococcus including Methicillin Resistant Staphaureus, streptococcal skin and oropharyngeal conditions, or gramnegative sepsis; a mycobacterial infection including tuberculosis; aviral infection including mononucleosis, Respiratory Syntitial virusinfection, or Herpes zoster infection; a parasitic infection includingmalaria, schistosomiasis, or trypanosomiasis.

Where the MDCMC is a chronic infection, it can be onchyomycosis; abacterial disorder including Helicobacter pylori; a mycobacterialinfection including tuberculosis; a viral infection including EpsteinBarr virus infection, Human Papilloma Virus infection, or Herpes SimplexVirus infection; or a parasitic infection including malaria orschistosomiasis.

In a fourteenth embodiment the present invention is directed acomposition that contains or is an implantable or attachable medicaldevice and an Fc reagent bound thereto.

In a fifteenth embodiment the present invention is directed a kit thatcontains an implantable or attachable medical device and an Fc reagent.In both these embodiments, the implantable or attachable medical deviceand the Fc reagent can be any of those recited herein. The kit canfurther contain a suitable container.

Additional advantages and features of the present invention will beapparent from the following detailed description, drawings and examples,which illustrate preferred embodiments of the invention.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows in schematic form a native Fc fragment monomer structurefrom IgG1 having a Hinge domain linked to a CH2 domain linked to a CH3domain; FIG. 1B shows a self-aggregated, native IgG1 Fc fragment formedfrom two associated Fc fragment monomers.

FIG. 1C shows in schematic form a native Fc fragment monomer structurefrom IgG3 having a Hinge domain linked to a CH2 domain linked to a CH3domain; FIG. 1D shows a self-aggregated, native IgG3 Fc fragment formedfrom two associated Fc fragment monomers.

FIGS. 2A and 2B show higher order aggregates of the native Fc fragmentstructure shown in FIG. 1B. Fc fragments may naturally multimerize intodimers of dimer (i.e. tetramers) or even higher order multimeraggregates.

FIG. 3A shows a schematic of a native IgG1 antibody having a native Fabfragment linked to the Fc fragment at the hinge of the Fc fragment; FIG.3B shows the analogous IgG3 structure.

FIG. 4A shows a stradomer monomer composed of two IgG1 Fc domainmonomers in series; FIG. 4B shows an alternative stradomer monomerstructure having linked in series IgG1 Fc-IgG3 Fc-IgE Fc.

FIGS. 5A & B show the stradomer monomers of FIGS. 4A & B autodimerizinginto a serial stradomer due to the intrinsic capacity of the componentFc domain monomers.

FIG. 6A shows a stradomer monomer containing IgG1 Fc-IgG1 (hinge-CH2);FIG. 6B shows a stradomer containing IgG1 (hinge-CH2)-IgG3(hinge-CH2)-IgE (hinge-CH2) derived sequences.

FIGS. 7A & B show the stradomer monomers of 6A & B autodimerizing into aserial stradomer due to the intrinsic capacity of the component Fcdomains.

FIG. 7C shows a serial stradomer containing IgE(hinge)-IgG1 Fc-IgG1(hinge-CH2)-IgE (CH3). FIG. 7D shows a serial stradomer containing anIgG3Fc-IgG1Fc.

FIG. 8A shows a stradobody construct containing a Fab with a serialstradomer structure with each stradomer monomer containing two IgG1CH2-CH3 derived Fc domain monomers; FIG. 8B shows a stradobody constructas in 8A but with a stradomer structure containing an IgG1 Fc linked toan IgG3 Fc linked to an IgE Fc.

FIG. 9A shows an IgG1 Fc-IgG1 (hinge-CH2) stradobody; FIG. 9B shows IgG1(hinge-CH2)-IgG3 (hinge-CH2)-IgE (hinge-CH2) 3-stradobody.

FIG. 10A shows an IgG1 (hinge-CH2)-IgG3 CH3-IgM CH4 stradomer monomerand a J chain protein; FIG. 10B shows a core stradomer based on afivemer of the stradomer of FIG. 10A formed by association through theIgM CH4 domain to a J chain.

FIG. 10C shows an IgG1 Fc-IgG1 Fc-IgM CH4 stradomer monomer and a Jchain protein; FIG. 10D shows a core stradomer based on a fivemer of thestradomer of FIG. 10C formed by association through the IgM CH4 domainto a J chain.

FIG. 11A shows an IgG1 Fc-IgG1 (hinge-CH2) stradomer monomer. FIG. 11Bdemonstrates how the stradomer monomer in FIG. 11A can auto-dimerize toform a serial stradomer. FIG. 11C demonstrates how the same stradomermonomer in FIG. 11A can have monomer Fc domains align with the same orsimilar Fc domain monomers on another stradomer monomer but not as anautodimer, thereby forming a stradomer composed of the same stradomermonomer as the autodimer but with a zipper effect structure.

FIG. 12A shows an IgG3 Fc-IgG1 Fc stradomer monomer. FIG. 12B shows thatthe addition of a second IgG3 Fc followed by autodimerization can form abranched structured IgG3 Fc-IgG1 Fc-IgG3 Fc stradomer.

FIG. 13A shows an IgE CH2-IgG1 Fc-IgG1 (hinge-CH2)-IgE CH4 stradomermonomer. FIG. 13B shows the autodimer of the FIG. 13A monomer andhighlights two FcγR binding sites formed.

FIG. 14A shows a stradomer composed of two IgG1 Fc domains joined by alinker. FIG. 14B shows a stradomer composed of two serial stradomers(specifically in each case a 2(IgG1 Fc) stradomer) joined by a linker.

FIG. 15A shows the nucleic acid (SEQ ID NO:1) and amino acid (SEQ IDNO:2) sequences of the human IgG1 Fc fragment. FIG. 15B shows thenucleic acid (SEQ ID NO:3) and amino acid (SEQ ID NO:4) sequences of thehuman IgG2 Fc fragment. FIG. 15C shows the nucleic acid (SEQ ID NO:5)and amino acid (SEQ ID NO:6) sequences of the human IgG3 Fc fragment.FIG. 15D shows the nucleic acid (SEQ ID NO:7) and amino acid (SEQ IDNO:8) sequences of the human IgG4 Fc fragment.

FIG. 16 shows the nucleic acid (SEQ ID NO:17) and amino acid (SEQ IDNO:18) sequences of a construct comprising {IgK signal sequence-IgG1 Fcfragment-IgG1 Fc fragment}. The amino acid sequence of the IgK signal isin bold. The amino acid sequence of the first IgG1 Fc fragment is singleunderlined. The amino acid sequence of the second IgG1 Fc fragment isdouble underlined. The serine and lysine marked with an asterisk arethose amino acids that may be mutated to alter Fcγ receptor binding.

FIG. 17 shows the nucleic acid (SEQ ID NO:19) and amino acid (SEQ IDNO:20) sequences of a construct comprising {Restriction Enzyme Sites-IgKsignal sequence-Restriction Enzyme Sites-IgG1(Hinge-CH2-CH3)-RestrictionEnzyme Sites-epitope tags(V5 and His)-STOP}. The amino acid sequence ofthe IgK signal is in bold. The amino acid sequence of the IgG1 Fcfragment is single underlined. The amino acid sequence of the V5 tag isunderlined with a dashed line. The amino acid sequence of the His tag isunderlined in bold.

FIG. 18 shows the nucleic acid (SEQ ID NO:21) and amino acid (SEQ IDNO:22) sequences of a construct comprising {Restriction Enzyme Sites-IgKsignal-Restriction Enzyme Sites-IgG1(Hinge-CH2-CH3)-XbaIsite-IgG1(Hinge-CH2-CH3)-STOP}. The amino acid sequence of the IgKsignal is in bold. The amino acid sequence of the first IgG1 Fc fragmentis single underlined. The amino acid sequence of the second IgG1 Fcfragment is double underlined.

FIG. 19 shows the nucleic acid (SEQ ID NO:23) and amino acid (SEQ IDNO:24) sequences of a construct comprising {Restriction Enzyme Sites-IgKsignal-Restriction Enzyme Sites-IgG1(Hinge-CH2-CH3)-XbaIsite-IgG1(Hinge-CH2-CH3)-Restriction Enzyme Sites-epitope tags(V5 andHis)-STOP}. The amino acid sequence of the IgK signal is in bold. Theamino acid sequence of the first IgG1 Fc fragment is single underlined.The amino acid sequence of the second IgG1 Fc fragment is doubleunderlined. The amino acid sequence of the V5 tag is underlined with adashed line. The amino acid sequence of the His tag is underlined inbold.

FIG. 20A shows the nucleic acid (SEQ ID NO:31) and amino acid (SEQ IDNO:32) sequences of the N-terminal signal sequence of FcRgammaIIIa withthe phenylalanine (F) polymorphism shown in bold and underlined. Thevariable nucleic acid is also in bold and underlined. FIG. 20B shows thenucleic acid (SEQ ID NO:33) and amino acid (SEQ ID NO:34) sequences ofthe N-terminal signal sequence of FcRgammaIIIa with valine (V)polymorphism shown in bold and underlined. The variable nucleic acid isalso in bold and underlined. Both constructs contain a C-terminalhexaHis tag for purification.

FIG. 21 shows the nucleic acid (SEQ ID NO:25) and amino acid (SEQ IDNO:26) sequences of a construct comprising {Restriction Enzyme Sites-IgKsignal-EcoRV Site-IgG3(Hinge-CH2-CH3)-IgG1(Hinge-CH2-CH3)-RestrictionEnzyme Sites-epitope tags(V5 and His)-STOP}. The amino acid sequence ofthe IgK signal is in bold. The amino acid sequence of the IgG3 Fcfragment is single underlined. The amino acid sequence of the IgG1 Fcfragment is double underlined. The amino acid sequence of the V5 tag isunderlined with a dashed line. The amino acid sequence of the His tag isunderlined in bold.

FIG. 22 shows the nucleic acid (SEQ ID NO:27) and amino acid (SEQ IDNO:28) sequences of a construct comprising {Restriction Enzyme Sites-IgKsignal-EcoRV Site-IgE(CH2)-IgG1(Hinge-CH2-CH3)-IgG1(Hinge-CH2)-IgE(CH4)-STOP}. The amino acidsequence of the IgK signal is in bold. The amino acid sequence of theIgE(CH2) domain is single underlined. The amino acid sequence of theIgG1(Hinge-CH2-CH3) domain is double underlined. The amino acid sequenceof the IgG1(Hinge-CH2) domain is underlined with a dashed line. Theamino acid sequence of the IgE (CH4) domain is underlined with a wavyline.

FIG. 23A shows an Fc fragment and demonstrates that such Fc fragment iscomposed of two Fc fragment monomers, and further comprises an Fc domain(dashed circle) and Fc partial domains (hinge, CH2 and CH3 asindicated). FIG. 23B shows the composition of a serial stradomer,composed of two stradomer monomers which are connected by aninter-stradomer monomer linkage. The serial stradomer comprises at leasttwo Fc domains (indicated as dashed circles) and may optionally comprisea domain linkage region. FIG. 23C shows the composition of a corestradomer comprising a core moiety to which are bound core stradomerunits that contain at least one Fc domain each. The core stradomer unitsmay be an Fc fragment, a serial stradomer or a cluster stradomer unit.FIG. 23D shows the composition of a cluster stradomer comprisingmultimerized cluster stradomer units, each of which has a multimerizingregion and a region containing at least one Fc domain. The clusterstradomer unit may be an Fc fragment or a serial stradomer. Themultimerizing region, once multimerized, forms the head of a clusterstradomer. The legs of the cluster stradomer are formed by the Fc domainregions of the cluster stradomer units that are spatially lessconstrained than the multimerized head of the cluster stradomer.

FIG. 24 shows the amino acid sequences of the stradomer set forth inTable 3.

FIG. 25 shows the amino acid sequences for the Fc partial domainsmonomers (hinge, CH2 and CH3) of human IgG1, IgG2, IgG3 and IgG4 (Kabat,E A, Wu, T T, Perry, H M, Gottesman, K S, and Foeller, C. 1991.Sequences of proteins of immunological interest 5th Ed. US Public HealthServices, NIH, Bethesda).

DETAILED DESCRIPTION OF THE INVENTION

The approach to rational molecular design for hIVIG replacementcompounds described herein includes recombinant and/or biochemicalcreation of immunologically active biomimetic(s). In preferred methods,these replacement compounds are screened in vitro to assess eachreplacement compound's efficiency at binding to Fcγ receptor andmodulating immune function. Particular replacement compounds areselected for further in vivo validation and dosage/administrationoptimization. The replacement compounds have utility for treating, forexample, autoimmune diseases, inflammatory diseases, osteoporosis, andcancer. Each phase is described in detail below along with specificexemplary embodiments.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.”

As used herein, the terms “biomimetic”, “biomimetic molecule”,“biomimetic compound”, and related terms, refer to a human made compoundthat imitates the function of another compound, such as pooled hIVIG, amonoclonal antibody or the Fc fragment of an antibody. “Biologicallyactive” biomimetics are compounds which possess biological activitiesthat are the same as or substantially similar to their naturallyoccurring counterparts. “Immunologically active” biomimetics arebiomimetics which exhibit immunological activity the same as orsubstantially similar to naturally occurring immunologically activemolecules, such as antibodies, cytokines, interleukins and otherimmunological molecules known in the art. In preferred embodiments, thebiomimetics of the present invention are stradomers and stradobodies, asdefined herein.

The immunologically active biomimetics of the present invention aredesigned to possess one or more immune modulating activities of the IgGFc domain and have at least (i) a first Fc domain capable of binding anFcγR, including FcγRI, FcγRII, FcγRIII and FcγRIV, and (ii) a second Fcdomain capable of binding an FcγR, including FcγRI, FcγRII, FcγRIII andFcγRIV.

The following paragraphs define the building blocks of the biomimeticsof the present invention, both structurally and functionally, and thendefine biomimetics themselves. However, it is first helpful to notethat, as indicated above, each of the biomimetics of the presentinvention has at least two Fc domains. At a minimum, an Fc domain is adimeric polypeptide (or a dimeric region of a larger polypeptide) thatcomprises two peptide chains or arms (monomers) that associate to form afunctional Fcγ receptor binding site. Therefore, the functional form ofthe individual fragments and domains discussed herein generally exist ina dimeric (or multimeric) form. The monomers of the individual fragmentsand domains discussed herein are the single chains or arms that mustassociate with a second chain or arm to form a functional dimericstructure.

Fc Fragment

“Fc fragment” is a term of art that is used to describe the proteinregion or protein folded structure that is routinely found at thecarboxy terminus of immunoglobulins (see FIG. 3A-3B). The Fc fragmentcan be isolated from the Fab fragment of a monoclonal antibody throughthe use of papain digestion, which is an incomplete and imperfectprocess (see Mihaesco C and Seligmann M. Papain Digestion Fragments OfHuman IgM Globulins. Journal of Experimental Medicine, Vol 127, 431-453(1968)). In conjunction with the Fab fragment (containing the antibodybinding domain) the Fc fragment constitutes the holo-antibody, meaninghere the complete antibody. The Fc fragment consists of the carboxyterminal portions of the antibody heavy chains. Each of the chains in anFc fragment is between about 220-265 amino acids in length and thechains are often linked via a disulfide bond. The Fc fragment oftencontains one or more independent structural folds or functionalsubdomains. In particular, the Fc fragment encompasses an Fc domain,defined herein as the minimum structure that binds an Fcγ receptor (see,e.g., FIGS. 1B and 1D). An isolated Fc fragment is comprised of two Fcfragment monomers (e.g., the two carboxy terminal portions of theantibody heavy chains; further defined herein) that are dimerized. Whentwo Fc fragment monomers associate, the resulting Fc fragment has Fcγreceptor binding activity.

Fc Partial Fragment

An “Fc partial fragment” is a domain comprising less than the entire Fcfragment of an antibody, yet which retains sufficient structure to havethe same activity as the Fc fragment, including Fcγ receptor bindingactivity. An Fc partial fragment may therefore lack part or all of ahinge region, part or all of a CH2 domain, part or all of a CH3 domain,and/or part or all of a CH4 domain, depending on the isotype of theantibody from which the Fc partial domain is derived. An example of a Fcpartial fragment includes a molecule comprising the upper, core andlower hinge regions plus the CH2 domain of IgG3 (Tan, L K, Shopes, R J,Oi, V T and Morrison, S L, Influence of the hinge region on complementactivation, C1q binding, and segmental flexibility in chimeric humanimmunoglobulins, Proc Natl Acad Sci USA. 1990 January; 87(1): 162-166).Thus, in this example the Fc partial fragment lacks the CH3 domainpresent in the Fc fragment of IgG3. Fc partial fragments are comprisedof two Fc partial fragment monomers. As further defined herein, when twosuch Fc partial fragment monomers associate, the resulting Fc partialfragment has Fcγ receptor binding activity.

Fc Domain

As used herein, “Fc domain” describes the minimum region (in the contextof a larger polypeptide) or smallest protein folded structure (in thecontext of an isolated protein) that can bind to or be bound by an Fcγreceptor. In both an Fc fragment and an Fc partial fragment, the Fcdomain is the minimum binding region that allows binding of the moleculeto an Fcγ receptor. While an Fc domain can be limited to a discretepolypeptide that is bound by an Fcγ receptor, it will also be clear thatan Fc domain can be a part or all of an Fc fragment, as well as part orall of an Fc partial fragment. When the term “Fc domains” is used inthis invention it will be recognized by a skilled artisan as meaningmore than one Fc domain. An Fc domain is comprised of two Fc domainmonomers. As further defined herein, when two such Fc domain monomersassociate, the resulting Fc domain has Fcγ receptor binding activity.Thus an Fc domain is a dimeric structure that functionally can bind anFcγ receptor.

Fc Partial Domain

As used herein, “Fc partial domain” describes a portion of an Fc domain.Fc partial domains include the individual heavy chain constant regiondomains (e.g., CH1, CH2, CH3 and CH4 domains) and hinge regions of thedifferent immunoglobulin classes and subclasses. Thus, Fc partialdomains of the present invention include the CH1 domains of IgG1, IgG2,IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE, the CH2 domains of IgG1, IgG2,IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE, the CH3 domains of IgG1, IgG2,IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE, the CH4 domains of IgM andIgE, and the hinge regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2,IgD and IgE. The Fc partial domain of the present invention may furthercomprise a combination of more than one more of these domains andhinges. However, the individual Fc partial domains of the presentinvention and combinations thereof lack the ability to bind an FcγR.Therefore, the Fc partial domains and combinations thereof comprise lessthan an Fc domain. Fc partial domains may be linked together to form apeptide that has Fcγ receptor binding activity, thus forming an Fcdomain. In the present invention, Fc partial domains are used with Fcdomains as the building blocks to create the biomimetics of the presentinvention, as defined herein. Each Fc partial domain is comprised of twoFc partial domain monomers. When two such Fc partial domain monomersassociate, an Fc partial domain is formed.

As indicated above, each of Fc fragments, Fc partial fragments, Fcdomains and Fc partial domains are dimeric proteins or domains. Thus,each of these molecules is comprised of two monomers that associate toform the dimeric protein or domain. While the characteristics andactivity of the dimeric forms was discussed above the monomeric peptidesare discussed as follows.

Fc Fragment Monomer

As used herein, an “Fc fragment monomer” is a single chain protein that,when associated with another Fc fragment monomer, comprises an Fcfragment. The Fc fragment monomer is thus the carboxy terminal portionof one of the antibody heavy chains that make up the Fc fragment of aholo-antibody (e.g., the contiguous portion of the heavy chain thatincludes the hinge region, CH2 domain and CH3 domain of IgG) (see FIG.1A and FIG. 1C)). In one embodiment, the Fc fragment monomer comprises,at a minimum, one chain of a hinge region (a hinge monomer), one chainof a CH2 domain (a CH2 domain monomer) and one chain of a CH3 domain (aCH3 domain monomer), contiguously linked to form a peptide. In anotherembodiment, the Fc fragment monomer comprises at least one chain of ahinge region, one chain of a CH2 domain, one chain of a CH3 domain, andone chain of a CH4 domain (a CH4 domain monomer) contiguously linked toform a peptide.

Fc Domain Monomer

As used herein, “Fc domain monomer” describes the single chain proteinthat, when associated with another Fc domain monomer, comprises an Fcdomain that can bind to an Fcγ receptor. The association of two Fcdomain monomers creates one Fc domain. An Fc domain monomer alone,comprising only one side of an Fc domain, cannot bind an Fcγ receptor.

Fc Partial Domain Monomer

As used herein, “Fc partial domain monomer” describes the single chainprotein that, when associated with another Fc partial domain monomer,comprises an Fc partial domain. The amino acid sequences of the Fcpartial domain hinge, CH2 and CH3 monomers for IgG1, IgG2, IgG3 and IgG4are shown in FIG. 25. The association of two Fc partial domain monomerscreates one Fc partial domain.

Stradomers

In particular embodiments, the biomimetics of the present inventioninclude stradomers. Stradomers are biomimetic compounds capable ofbinding two or more Fcγ receptors (see, e.g., FIG. 13B). In a preferredembodiment, the stradomers of the present invention are used to bind Fcγreceptors on effector cells such as NK cells and immature dendriticcells and other monocyte-derived cells. In one embodiment, the Fcγreceptors are low affinity Fcγ receptors. A stradomer can have fourdifferent physical conformations: serial, cluster, core or Fc fragment,each of which is discussed in the following paragraphs. As will beevident, the Fc fragments, Fc partial fragments, Fc domains and Fcpartial domains discussed above are used in the construction of thevarious stradomer conformations. Further, it is the individual Fc domainmonomers and Fc partial domain monomers, also discussed above, that arefirst produced, and that then self-associate to form the dimericstructures that are the stradomers of the present invention.

Serial Stradomer

A “serial stradomer” is dimeric polypeptide comprised of two linearstradomer monomers that, when associated, form two or more Fc domains.The Fc domains of the stradomer are only functional when the two peptidechains (stradomer monomers) are associated (i.e., non-functional in themonomeric state). Thus a serial stradomer is a biomimetic compoundcapable of binding two or more Fcγ receptors. In different embodiments,serial stradomer may have two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen or more Fc domains, aswell as Fc partial domains. The Fc domains, and Fc partial domains,within a serial stradomer may be linked by domain linkages, as furtherdefined herein.

As used herein, a “stradomer dimer” is a specific form of a stradomer,composed of only two stradomers. In one embodiment, the stradomer dimersare molecules formed by self-aggregation of relevant stradomer monomers.In another embodiment, stradomer monomers in the stradomer dimers arephysically linked through an inter-stradomer monomer linkage, as definedherein. A “multimeric stradomer” is comprised of three or morestradomers, formed by self-aggregation of stradomer monomers, or throughan inter-stradomer monomer linkage, as defined herein in.

Stradomer Monomer

As used herein, the term “stradomer monomer” refers to a single,contiguous peptide molecule that, when associated with at least a secondstradomer monomer, forms a polypeptide comprising at least two Fcdomains (see, e.g., FIGS. 6A-6B, FIG. 12A). While in preferredembodiments serial stradomer are comprised of two associated stradomermonomers (see, for example, FIGS. 5A, 5B, 7A, 7B, 7C, 7D), a serialstradomer may also contain three (see FIG. 11C) or more stradomermonomers. Stradomer monomers may be associated to form stradomers byinter-stradomer monomer linkages or they may form stradomers throughself-aggregation.

A stradomer monomer may have an amino acid sequence that will form one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or more Fc domains when associated with anotherstradomer monomer to form a stradomer. A stradomer monomer may furtherhave an amino acid sequence that will form one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or moreFc partial domains when associated with another stradomer monomer toform a stradomer.

The regions of stradomer monomers that will form Fc domains and Fcpartial domains in the context of a stradomer may simply be arrangedfrom carboxy terminal to amino terminal of successive regions of thestradomer monomer molecule (see, e.g., FIG. 4A-4B). Alternatively, thesuccessive regions of the stradomer monomers may be linked through apeptide sequence termed a “domain linkage” herein. The arrangement ofthe particular Fc domain monomers and Fc partial domain monomerscomprising a stradomer monomer is not critical. However, the arrangementmust permit formation of two functional Fc domains upon association oftwo stradomer monomers.

In one embodiment of the stradomers of the present invention, stradomermonomers are produced that contain at the N-terminus of the peptide anFc domain monomer or Fc partial domain monomer that binds strongly toitself, such as a single or two terminal IgE CH2 domain monomers or apartial IgG3 hinge domain monomer, to create an Fc domain or an Fcpartial domain, respectively. Each of these stradomer monomers has therequisite complement of Fc domain monomers and/or partial Fc domainmonomers to bind to two Fc gamma receptors upon formation of astradomer. Stradomers that result from association of such stradomermonomers are biomimetics capable of binding two or more Fc gammareceptors. In a preferred embodiment the N-terminal Fc domain or Fcpartial domain contains an additional glycosylation site such as thatwhich exists on the IgE CH2 domain.

As a clarifying example, the skilled artisan will understand that thestradomer molecules of the present invention may be constructed bypreparing a polynucleotide molecule that encodes various combinations ofFc domain monomers and Fc partial domain monomers, but with acombination that will form a minimum of two Fc domain monomers. Such apolynucleotide molecule may be inserted into an expression vector, whichcan be used to transform a population of bacteria. Stradomer monomerscan then produced by culturing the transformed bacteria underappropriate culture conditions. Stradomer monomers can then formfunctional stradomers upon either self-aggregation of the stradomermonomers or association of stradomer monomers using inter-stradomermonomer linkages. The present invention encompasses both stradomersformed through the association of stradomer monomers having identicalamino acid sequences, stradomer monomers having substantially similaramino acid sequences, or stradomer monomers having dissimilar sequences.In the latter embodiment the amino acid sequence of the stradomermonomers comprising a stradomer need only be of such similarity that twoor more functional Fcγ receptor binding sites are formed.

As indicated above, an Fc domain can be functionally defined by itsability to bind an Fcγ receptor. As a result, the particular amino acidsequence of an Fc domain will vary based on the Fc partial domains thatcomprise the Fc domain. However, in one embodiment of the presentinvention the Fc domain comprises the hinge region and a CH2 domain ofan immunoglobulin molecule. In a further embodiment the Fc domaincomprises the hinge region, a CH2 domain and CH3 domain of animmunoglobulin molecule. In a further embodiment, the Fc domaincomprises the hinge region, a CH2 domain, CH3 domain and CH4 domain ofan immunoglobulin molecule. In yet another embodiment, the Fc domaincomprises the hinge region, a CH2 domain and CH4 domain of animmunoglobulin molecule.

Domain Linkage

As indicated above, a “domain linkage” is a peptide linkage between Fcdomain monomers and/or Fc partial domain monomers that comprise each ofthe individual stradomer monomers of the serial stradomers orstradobodies of the present invention. The domain linkage may be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moreamino acids. A domain linkage does not occur between Fc partial domainmonomers that are in their natural sequence. That is, where linkednaturally contiguous portions of Fc domain monomers are used, such asthe hinge region, CH2 domain and CH3 domain of IgG, these Fc partialdomain monomers comprise a contiguous sequence and no domain linkagebetween these elements is required. In contrast, for example, when twoor more Fc domain monomers or partial Fc domain monomers are linked in amanner that is not naturally occurring to form an individual stradomermonomer, domain linkages may be used. An example would be the linkagebetween two hinge/CH2/CH3 peptides, creating an individual stradomermonomer of a stradomer comprising: hinge/CH2/CH3/L/hinge/CH2/CH3, where“L” is the domain linkage (see, e.g., FIG. 4A where the domain linkage(not shown) occurs between the IgG1 CH3 domain and the IgG1 hinge). Inthe various cases described, the domain linkage may be one of thenaturally occurring portions of the heavy chain that joins the hinge andCH domains in the Fc domain monomer of an antibody. Alternatively, thedomain linkage may be any other amino acid sequence that provides neededspacing and flexibility between the Fc domain monomers and partial Fcdomain monomers of an individual stradomer monomer and that allows theindividual stradomer monomers to pair with other each other to form thestradomers of the present invention.

The skilled artisan will understand that the identity of the domainlinkage is not particularly important as long as it permits two or moreindividual stradomer monomers to form the biomimetic compounds of thepresent invention, and that the resulting compounds have the ability tocross-link more than one FcγR. It is envisioned that eachimmunologically active biomimetic compound will preferably contain atleast one domain linkage in each stradomer monomer of the serialstradomer or stradobody which will function to maintain the Fc domainsof the immunologically active biomimetic within a restricted spatialregion and which will facilitate FcγR activation activity, for example,by aggregating FcγRs through co-binding to the Fc domains within theimmunologically active biomimetic. Preferably, the domain linkages willallow the same or a greater degree of conformational variability as isprovided by the hinge domain of IgG molecules. All the above linkagesare well-known in the art.

Inter-Stradomer Monomer Linkage

A separate linkage found in the biomimetic compounds of the presentinvention is the “inter-stradomer monomer linkage” that occurs betweentwo or more individual stradomer monomers that comprise the stradomersand stradobodies of the present invention. While the domain linkages areshort amino acid sequences that serve to link the Fc domain monomers andpartial Fc domain monomers that comprise individual stradomer monomersof the biomimetic compounds to each other, the inter-stradomer monomerlinkages serve to join two or more individual stradomer monomers thatcomprise the biomimetic compounds. The inter-stradomer monomer linkagemay be any linkage capable of stably associating the individualstradomer monomers. In some embodiments, the inter-stradomer monomerlinkage may be a covalent link between the stradomer monomers.Alternatively, the inter-stradomer monomer linkage between stradomermonomers may be by direct chemical crosslinking. In preferredembodiments, the stradomer monomer structures take advantage of thenatural self-aggregation properties between Fc domain monomers to createself-aggregating stradomers. In such embodiments, disulfide bonds formbetween the individual stradomer monomers to form the stradomers (see,e.g., FIG. 5A, where inter-stradomer monomer linkages (not shown) serveto join the two individual stradomer monomers of the stradomer). Thedisulfide bonds form between cysteine residues of the Fc domain monomersthat comprise the biomimetic molecules, using either cysteine residuesoccurring in the natural Fc domain monomer sequence or cysteine residuesincorporated into an Fc domain monomer by site-directed mutagenesis.Such natural self-aggregation properties can also be used to form theinter-stradomer monomer linkages between individual stradomer monomersin stradomer multimers. Alternative embodiments include inter-stradomermonomer linkages where disulfide bonds form between cysteine residuesintroduced through site-directed mutagenesis into the amino acidsequence comprising the individual stradomer monomers.

As discussed above, in a preferred embodiment, the inter-stradomermonomer linkage that forms a stradomer is a linkage that results fromself-aggregation of stradomer monomers. In one embodiment, the twostradomer monomers that comprise the stradomer are identical peptides,such that the two individual stradomer monomers that comprise thestradomer are identical in sequence. However, the skilled artisan willunderstand that other embodiments include stradomers where the stradomermonomers differ from each other in amino acid sequence.

Two stradomer monomers can form a stradomer by, for example, aligning inparallel such that pairing takes place between identical Fc partialdomain monomers in the stradomer monomers (see, e.g., FIGS. 5A-B).However, the present invention also includes embodiments where pairingoccurs between non-identical Fc partial domain monomers, and embodiments(see FIG. 11C) where pairing occurs between identical Fc partial domainmonomers in the stradomer monomers but where the alignment of the twostradomer monomers is offset.

In order to control the production and self-dimerization of a stradomermonomer, “capping regions” may be used. For example, a stradomer monomersequence may comprise the following Fc partial domains: IgE CH2/IgG1hinge/IgG1 CH2/IgG1 CH3/IgG1 hinge/IgG1 CH2/IgE CH4, (see FIG. 13A)where the IgE domains serve as a cap to prevent a “zippering effect.” Azippering effect can occur when a stradomer monomer (see FIG. 11A) canauto-dimerize (see FIG. 11B) or can align itself not as an auto-dimerbut as alternating monomers in parallel (see FIG. 11C). One of ordinaryskill in the art will understand that a variety of Fc partial domains,such as the hinge of any immunoglobulin or the CH4 domain of IgM or IgE,may be used alone or in combination to direct the stradomer toauto-dimerize and to prohibit the zippering effect when desired. Othernon-series structures may contain branched molecules (see FIG. 12B), twoor more stradomers lined up in parallel joined by linkers such as asimple covalent bond, peptide linkers, or non-peptide linkers (see FIGS.14A and 14B).

Core Stradomer

A “core stradomer” is comprised of a core moiety to which are bound twoor more core stradomer units, wherein each core stradomer unit comprisesat least one Fc domain, thereby creating a biomimetic compound capableof binding two or more Fcγ receptors. An Fc fragment, Fc partialfragment, serial stradomer or cluster stradomer unit can eachindependently serve as one or both (if they comprise two Fc domains) ofthe core stradomer units in a core stradomer because each of thesemolecules contains at least one Fc domain. Thus, a core stradomer maycomprise a core moiety to which is bound at least one serial stradomer.

As used herein, the core moiety of a core stradomer is any physicalstructure to which the core stradomer units may be linked or covalentlybound. Preferred polypeptides that may serve as the core moiety includekeyhole limpet hemocyanin, bovine serum albumin and ovalbumin. Chemicalcrosslinking between such core moieties and core stradomer units (e.g.,Fc fragment, Fc partial fragment, Fc domain, serial stradomer andcluster stradomer unit) may be achieved by means of numerous chemicalsusing well known techniques. Exemplary chemicals generally suitable foruse in the crosslinking include glutaraldehyde, carbodiimide,succinimide esters (e.g. MBS, SMCC), benzidine, periodate,isothiocyanate; PEO (polyethylene)/PEG (polyethylene glycol) spacerssuch as Bis(NHS)PEO₅, DFDNB (1,5-Difluoro-2,4-dinitrobenzene); and AmineReactive homobifunctional cross-linking reagents includingAldehyde-Activated Dextran, Bis(Sulfosuccinimidyl)suberate,Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, Dimethyl adipimidate.2HCl, Dimethyl pimelimidate.2 HCl, Dimethyl Suberimidate.2 HCl,Disuccinimidyl glutarate, Dithiobis(succinimidyl) propionate,Disuccinimidyl suberate, Disuccinimidyl tartrate, Dimethyl3,3′-dithiobispropionimidate.2 HCl,3,3′-Dithiobis(sulfosuccinimidylpropionate), Ethylene glycolbis[succinimidylsuccinate], Ethylene glycolbis[sulfosuccinimidylsuccinate],β-[Tris(hydroxymethyl)phosphino]propionic acid and Tris-succinimidylaminotriacetate. One of skill in the art will be able to select theappropriate crosslinking chemical and conditions based upon theparticular core moiety selected and the sequence of the Fcdomain-containing polypeptides being combined to form an immunologicallyactive biomimetic. See, e.g., Wong, Shan S. Chemistry of proteinconjugation and cross-linking Boca Raton: CRC Press, c1991 (ISBN0849358868).

In another preferred embodiment, a joining (J) chain polypeptide may beused as the core moiety. When a J chain is used as the core moiety,cysteine bridges may be used to connect individual core stradomer unitsto form a core stradomer (See FIG. 10A-10D). In an embodiment of a corestradomer, serial stradomers (serving as the core stradomer units)containing a terminal IgM CH4 domain are associated with a J chain toform a core stradomer. The inclusion of the IgM CH4 domain results inthe self-aggregation of stradomers comprising this Fc partial domainwith a J chain to form a biomimetic capable of binding multiple Fc gammareceptors. Another exemplary core stradomer is one comprising Fc domains(serving as the core stradomer units) where the Fc domains have thestructure IgG3 hinge/IgG3 CH2/IgG3 CH3/IgM CH4. The component Fc domainsof this molecule cannot individually bind more than one Fc gammareceptor, but the entire structure can bind five Fc gamma receptors whenthe component Fc domains associate with a J chain.

In another embodiment, the core moiety may be a non-polypeptide entity.A variety of suitable compositions may be physically associated with thecore stradomer units to produce an immunologically active biomimetic.Non-toxic beads, hyperbranched polymers and dendrimers, nanoparticles,and various compounds that are classified by the FDA as GenerallyRegarded As Safe (e.g. propylene glycol, sorbitol, liposomes andsilicate calcium) may be used. See, e.g., Nanoparticulates as DrugCarriers by Vladimir P. Torchilin (Editor), Imperial College Press(September 2006) ISBN: 1860946305/ISBN-13: 9781860946301.

Preferred core moieties of the present invention include a bead,albumin, a liposome, a peptide and polyethylene glycol.

Cluster Stradomer

A “cluster stradomer” is a biomimetic that has an octopus-like form witha central moiety “head” and two or more “legs”, wherein each legcomprises one or more Fc domain that is capable of binding at least oneFc gamma receptor, thus creating a biomimetic capable of binding two ormore Fc gamma receptors. Each cluster stradomer is comprised of morethan one dimeric protein, each called a “cluster stradomer unit.” Eachcluster stradomer unit is comprised of a region that multimerizes and a“leg” region that comprises at least one functional Fc domain. Themultimerizing region creates a cluster stradomer “head” oncemultimerized with the multimerizing region of another cluster stradomerunit. The leg region is capable of binding as many Fcγ receptors asthere are Fc domains in each leg region. Thus a cluster stradomer is abiomimetic compound capable of binding two or more Fcγ receptors.

The multimerizing region may be a peptide sequence that causes dimericproteins to further multimerize or alternatively the multimerizingregion may be a glycosylation that enhances the multimerization ofdimeric proteins. Examples of peptide multimerizing regions include IgG2hinge, IgE CH2 domain, isoleucine zipper, and zinc fingers. Theinfluence of glycosylation on peptide multimerization is well describedin the art (e.g., Role of Carbohydrate in Multimeric Structure of FactorVIII/V on Willebrand Factor Protein. Harvey R. Gralnick, Sybil B.Williams and Margaret E. Rick. Proceedings of the National Academy ofSciences of the United States of America, Vol. 80, No. 9, [Part 1:Biological Sciences] (May 1, 1983), pp. 2771-2774; Multimerization andcollagen binding of vitronectin is modulated by its glycosylation. KimieAsanuma, Fumio Arisaka and Haruko Ogawa. International Congress SeriesVolume 1223, December 2001, Pages 97-101).

A trained artisan will recognize that a cluster stradomer unit mayitself comprise a serial stradomer (containing two or more Fc domains)along with a multimerizing region. Thus the “legs” of a clusterstradomer may be comprised of any of the types of serial stradomersdiscussed herein and/or one or more of an IgG1 Fc fragment and/or anIgG3 Fc fragment and/or a single Fc domain. One trained in the art willrecognize that each of the IgG1 Fc fragments and IgG3 Fc fragment insuch biomimetics may be modified to comprise partial Fc fragments fromany immunoglobulin. The monomers that comprise the cluster stradomerunit (which, as indicated above, exists as a dimeric association of twopeptides) are “cluster stradomer unit monomers.” An exemplary clusterstradomer that has been made whose cluster stradomer unit would not bindmore than one low affinity Fc gamma receptor prior to multimerizationis: IgE CH2/IgG1 hinge/IgG1 CH2/IgG1 CH3.

One trained in the art will recognize that when a serial stradomer isused as the “leg” of a cluster stradomer, each “leg” will be capable ofbinding more than one Fc gamma receptor (as at least two Fc domains arepresent in a serial stradomer), thus creating a biomimetic capable ofbinding more than one Fc gamma receptor. Fc partial domains, otherimmunoglobulin sequences, and non-immunoglobulin sequences may be placedat the termini of individual cluster stradomer unit monomers comprisingthe legs to create a cluster stradomer wherein each leg has preferredspatial proximity to increase their availability to bind one or morethan one Fc gamma receptor.

The multimerizing region may be a peptide sequence that causes peptidesto dimerize or multimerize and includes the IgG2 hinge, the IgE CH2domain, an isoleucine zipper and a zinc finger. As is known in the art,the hinge region of human IgG2 can form covalent dimers (Yoo, E. M. etal. J. Immunol. 170, 3134-3138 (2003); Salfeld Nature Biotech. 25,1369-1372 (2007)). The dimer formation of IgG2 is potentially mediatedthrough the IgG2 hinge structure by C—C bonds (Yoo et al 2003),suggesting that the hinge structure alone can mediate dimer formation.Thus, serial stradomers having an IgG2 hinge (and thus serving ascluster stradomer units) will form a cluster stradomer that may comprisetwo serial stradomers or even three serial stradomers.

The amino acid sequence of the human IgG2 hinge monomer is as follows:ERKCCVECPPCP (SEQ ID NO: 36). The core structure of the hinge is theC-X-X-C portion of the hinge monomer. Thus, stradomer monomers of thepresent invention may comprise either the complete 12 amino acidsequence of the IgG2 hinge monomer, or the four amino acid core, alongwith Fc domain monomers. While the X-X of the core structure can be anyamino acid, in a preferred embodiment the X-X sequence is V-E or P-P.The skilled artisan will understand that the IgG2 hinge monomer may becomprised of any portion of the hinge sequence in addition to the corefour amino acid structure, including all of the IgG2 hinge sequence andsome or all of the IgG2 CH2 and CH3 domain monomer sequences. Specificexamples of possible IgG2 hinge-IgG1 Fc domain serial stradomerconstructs are as follows:

TABLE 1 N-term H CH2 CH3 H CH2 CH3 H CH2 CH3 C-term CXXC 1 1 1 CXXC 1 11 1 1 1 2 2 2 1 1 1 2 2 2 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 2x 2 2 1 1 12x 2 2 1 1 1 1 1 1 2x 2 2 1 1 1 1 1 1 IgE hinge 2x 1 1 1 Nomenclature: H= hinge, CH2 = constant heavy domain 2, CH3 = constant heavy domain 3, 1= IgG1, 2 = IgG2, X = any amino acid; 2x = two hinges in consecutiveorder

These are only a few of many examples. Any of the IgG1 Fc domains can,for example, be replaced with an IgG3 Fc domain. Additional proteinswith IgG2 dimerization domains includes IgG2-IgG1 chimeric proteins withthe addition of N and/or C terminal sequences comprising IgM or IgEdomain monomer sequences. These N and C terminal sequences can be hingeregions, constant domains, or both.

As indicated above, leucine and isoleucine zippers may also be used asthe multimerizing region. Leucine and isoleucine zippers (coiled-coildomains) are known to facilitate formation of protein dimers, trimersand tetramers (Harbury et al. Science 262:1401-1407 (1993); O'Shea etal. Science 243:538 (1989)). By taking advantage of the natural tendencyof an isoleucine zipper to form a trimer, cluster stradomers may beproduced using serial stradomers comprising an isoleucine zipper.Association of three or more serial stradomers (as cluster stradomerunits) having isoleucine zippers results in the formation of clusterstradomers having at least six Fc gamma receptor binding regions.

While the skilled artisan will understand that different types ofleucine and isoleucine zippers may be used, in a preferred embodimentthe isoleucine zipper from the GCN4 transcriptional regulator modifiedas described (Morris et al., Mol. Immunol. 44:3112-3121 (2007); Harburyet al. Science 262:1401-1407 (1993)) is used:YTQKSLSLSPGKELLGGGSIKQIEDKIEEILSKIYHIENEIARIKKLIGERGHGGGSNSQVSHRYPRFQSIKVQFTEYKKEKGFILTS (SEQ ID NO:37) This isoleucine zippersequence is only one of several possible sequences that can be used formultimerization of Fc domain monomers. While the entire sequence shownin SEQ ID NO:37 may be used, the underlined portion of the sequencerepresents the core sequence of the isoleucine zipper that may be usedin the cluster stradomers of the present invention. Thus, stradomermonomers of the present invention may comprise either the complete 88amino acid sequence of the isoleucine zipper (ILZ), or the 28 amino acidcore, along with one or more Fc domain monomers. The skilled artisanwill also understand that the isoleucine zipper may be comprised of anyportion of the zipper in addition to the core 28 amino acid structure,and thus may be comprised of more than 28 amino acids, but less than 88amino acids of the isoleucine zipper. Specific examples of possibleILZ-IgG1 Fc domain constructs are shown as follows.

TABLE 2 H CH2 CH3 H CH2 CH3 ILZ 1 1 1 ILZ 1 1 1 1 1 ILZ 1 1 1 1 1 1 ILZ1 1 1 3 3 3 Nomenclature: H = hinge, CH2 = constant heavy domain 2, CH3= constant heavy domain 3, 1 = IgG1, 3 = IgG3, ILZ = isoleucine zipperdomain

These are only a few of many examples. Any of the IgG1 domains can, forexample, be replaced with IgG3 domains. Additional proteins with ILZdomains include IgG1 chimeric proteins with the addition of N and/or Cterminal sequences from other Ig molecules like IgM or IgE. These N andC terminal sequences can be hinge regions, constant domains or both.

Fc Fragment Stradomer

An “Fc fragment stradomer” is comprised of more than one Fc fragment.Under certain circumstances attributable to post-translationalmodification of the Fc fragment, the Fc fragment binds with sufficientstrength to another Fc fragment to permit the formation of a moleculethat binds to more than one Fcγ receptor. The post-translationalmodification that permits such binding includes glycosylation andmethylation. The identity of the cell line in which the recombinant Fcfragments are produced, and conditions under which they are produced,govern whether Fc fragments will form Fc fragment stradomers. Forexample, a recombinant Fc fragment produced in a FreestyleMax CHOtransient transfection cell forms multimers that are visible on westernblots, binds according to a bivalent fit on plasmon resonance bindingassay, and demonstrates biological activity in a dendritic cell assaycomparable to IVIG. In contrast, the same recombinant Fc fragmentproduced in a stable CHO cell line does not form multimers of the Fcfragment on western blots, binds according to a univalent fit on Plasmonresonance binding assay, and does not demonstrate comparable biologicalactivity. Thus an Fc fragment stradomer is a biomimetic compound capableof binding two or more Fcγ receptors.

As also used herein, the term “Fc dimer” is a dimer of Fc fragments (seeFIG. 2A), the term “Fc trimer” is a trimer of Fc fragments, and the term“Fc multimer” is a multimer of Fc fragments (see FIG. 2B).

Stradobody

The present invention also encompasses stradobodies. As used herein,“stradobody” refers to a molecule comprising two or more Fc domains,preferably in the context of a stradomer (including serial stradomers,core stradomers, cluster stradomers and Fc fragment stradomers), towhich one or more Fab domains is attached (see, e.g., FIGS. 8A-B and9A-B). Thus, by virtue of such Fab domains, stradobodies have bothantigen binding capacity, as well as stradomer Fcγ receptor bindingactivity. In some embodiments, the Fcγ receptor binding activity may bedue to an ability to bind and cross-link FcγR equal to or greater thanthe Fc portion of a native structure holo-antibody. Preferably the Fabportion of the stradobody comprises both a heavy and a light chain. Thevariable heavy chain and the light chain may be independently from anycompatible immunoglobulin such as IgA1, IgA2, IgM, IgD, IgE, IgG1, IgG2,IgG3, or IgG4, and may be from the same or different Ig isotype, butpreferably are from the same Ig isotype. The light chains kappa orlambda may also be from different Ig isotypes. Stradobodies, likestradomers, can bind two or more FCγRs and modulate immune function.

In one embodiment, the stradomers may have a Fab of an immunoglobulinlinked to an Fc hinge (H) domain of a stradomer to generate a stradobody(e.g. FIGS. 8A & B). In another embodiment, the stradobody may becomprised of IgG1 Fc-IgG1 (hinge-CH2) (e.g., FIG. 9A). In otherembodiments, the stradobody may be comprised of an IgG1 domain andhinge, an IgG3 domain and hinge and an IgGE domain and hinge (e.g., FIG.9B). The Fab comprises both a heavy and a light chain as found in nativeimmunoglobulin structures (FIG. 3A-B).

Stradobodies will possess the antigen binding properties of the Fabportion and the above described stradomer properties. Such a combinationwill serve to bind, cross-link, and activate Fcγ receptors on effectorcells at a higher rate than can be accomplished by an Fc backbone of aholo-antibody, particularly in the environment of low epitope expression(e.g. the 90% of breast cancer patients whose tumors are not classifiedas her/2-neu high expressors), inducing ADCC in a higher percentage ofpatients. As indicated above, one or more antigen-binding Fab fragmentscan be added to the stradomers to form stradobodies. Preferably,polypeptides (other than the linkages described herein) added tostradomers are not all or parts of non-immunoglobulin polypeptides.

The Fab may be a chimeric structure comprised of human constant regionsand non-human variable regions such as the variable region from a mouse,rat, rabbit, monkey, or goat antibody. One of ordinary skill in the artwould be able to make a variety of Fab chimeric structures forincorporation into stradobodies using methodologies currently availableand described in the scientific literature for such constructions. Thus,“humanized” stradobodies may be designed analogous to “humanizedmonoclonal antibodies.

Variants and Homologs

The skilled artisan will understand that the stradomers and otherbiomimetics of the present invention can be designed to include specificimmunoglobulin Fc domains, such as two Fc domains from IgG1 (i.e., IgG1hinge/IgG1 CH2/IgG1 CH3/IgG1 hinge/IgG1 CH2/IgG1 CH3). Such a stradomercould be constructed by first preparing a polynucleotide encoding twoIgG1 Fc domain monomers (i.e., IgG1 hinge monomer/IgG1 CH2 monomer/IgG1CH3 monomer/IgG1 hinge monomer/IgG1 CH2 monomer/IgG1 CH3 monomer), andthen expressing stradomer monomers there from. Upon association of twosuch stradomer monomers a serial stradomer having two IgG1 Fc domainswould be produced.

The stradomers and other biomimetics of the present invention can alsobe designed based on the identity of specific immunoglobulin Fc partialdomains that comprise the Fc domains. For example, a serial stradomercould be produced having two Fc domains, where the first Fc domaincomprises IgG1 hinge/IgG3 CH2/IgG1 CH3 and the second Fc domaincomprises IgG3 hinge/IgG1 CH2/IgG3 CH3.

It is understood that the stradomers and other biomimetic moleculesdisclosed herein can be derived from any of a variety of species.Indeed, Fc domains, or Fc partial domains, in any one biomimeticmolecules of the present invention can be derived from immunoglobulinfrom more than one (e.g., from two, three, four, five, or more) species.However, they will more commonly be derived from a single species. Inaddition, it will be appreciated that any of the methods disclosedherein (e.g., methods of treatment) can be applied to any species.Generally, the components of a biomimetic applied to a species ofinterest will all be derived from that species. However, biomimetics inwhich all the components are of a different species or are from morethan one species (including or not including the species to which therelevant method is applied) can also be used.

The specific CH1, CH2, CH3 and CH4 domains and hinge regions thatcomprise the Fc domains and Fc partial domains of the stradomers andother biomimetics of the present invention may be independentlyselected, both in terms of the immunoglobulin subclass, as well as inthe organism, from which they are derived. Accordingly, the stradomersand other biomimetics disclosed herein may comprise Fc domains andpartial Fc domains that independently come from various immunoglobulintypes such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM.Similarly each Fc domain and partial Fc domain may be derived fromvarious species, preferably a mammalian species, including non-humanprimates (e.g., monkeys, baboons, and chimpanzees), humans, murine,rattus, bovine, equine, feline, canine, porcine, rabbits, goats, deer,sheep, ferrets, gerbils, guinea pigs, hamsters, bats, birds (e.g.,chickens, turkeys, and ducks), fish and reptiles to producespecies-specific or chimeric stradomer molecules.

The individual Fc domains and partial Fc domains may also be humanized.One of skill in the art will realize that different Fc domains andpartial Fc domains will provide different types of functionalities. Forexample, FcγRs bind specifically to IgG immunoglobulins and not otherclasses of immunoglobulins. Thus, one of skill in the art, intending todesign a stradomer with multiple Fcγ receptor binding capacity, woulddesign stradomer Fc domains that at least incorporate the wellcharacterized Fcγ receptor binding sequences of IgG, including those inthe IgG hinge region and the IgG CH2 & CH3 domains. One of ordinaryskill in the art will also understand various deleterious consequencescan be associated with the use of particular Ig domains, such as theanaphylaxis associated with IgA infusions. The biomimetics disclosedherein should generally be designed to avoid such effects, although inparticular circumstances such effects may be desirable.

The present invention also encompasses stradomers comprising Fc domainsand Fc partial domains having amino acids that differ from thenaturally-occurring amino acid sequence of the Fc domain or Fc partialdomain. Preferred Fc domains for inclusion in the biomimetic compoundsof the present invention have a measurable specific binding affinity toeither a holo-Fcγ receptor or a soluble extracellular domain portion ofan FcγR. Primary amino acid sequences and X-ray crystallographystructures of numerous Fc domains and Fc domain monomers are availablein the art. See, e.g., Woof J M, Burton D R. Human antibody-Fc receptorinteractions illuminated by crystal structures. Nat Rev Immunol. 2004February; 4(2):89-99. Representative Fc domains with Fcγ receptorbinding capacity include the Fc domains from human immunoglobulin Gisotypes 1-4 (hIgG₁₋₄) (SEQ ID NOS: 1, 3, 5 and 7 respectively; see alsoFIG. 15A-D). (See FIG. 2 of Robert L. Shields, et al. High ResolutionMapping of the Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII,and FcRn and Design of IgG1 Variants with Improved Binding to the FcγR.J. Biol. Chem., February 2001; 276: 6591-6604). These native sequenceshave been subjected to extensive structure-function analysis includingsite directed mutagenesis mapping of functional sequences¹⁴. Based onthese prior structure-function studies and the available crystallographydata, one of skill in the art may design functional Fc domain sequencevariants (e.g., of SEQ ID NOS: 1, 3, 5 and 7) while preserving the Fcdomain's Fcγ receptor binding capacity.

The amino acid changes may be found throughout the sequence of the Fcdomain, or be isolated to particular Fc partial domains that comprisethe Fc domain. The functional variants of the Fc domain used in thestradomers and other biomimetics of the present invention will have atleast about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity to a native Fc domain. Similarly, the functional variants ofthe Fc partial domains used in the stradomers and other biomimetics ofthe present invention will have at least about 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98% or 99% sequence identity to a native Fc partialdomain.

The skilled artisan will appreciate that the present invention furtherencompasses the use of functional variants of Fc domain monomers in theconstruction of Fc fragment monomers, Fc partial fragment monomers,stradomer monomers and the other monomers of the present invention. Thefunctional variants of the Fc domain monomers will have at least about50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity toa native Fc domain monomer sequence.

Similarly, the present invention also encompasses the use of functionalvariants of Fc partial domain monomers in the construction of Fcfragment monomers, Fc partial fragment monomers, Fc domains monomers,stradomer monomers and the other monomers of the present invention. Thefunctional variants of the Fc partial domain monomers will have at leastabout 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity to a native Fc partial domain monomer sequence.

The amino acid changes may decrease, increase, or leave unaltered thebinding affinity of the stradomer to the Fcγ receptor. Preferably suchamino acid changes will be conservative amino acid substitutions,however, such changes include deletions, additions and othersubstitutions. Conservative amino acid substitutions typically includechanges within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine,glutamine, serine and threonine; lysine, histidine and arginine; andphenylalanine and tyrosine.

The term “functional variant” as used herein refers to a sequencerelated by homology to a reference sequence which is capable ofmediating the same biological effects as the reference sequence (when apolypeptide), or which encodes a polypeptide that is capable ofmediating the same biological effects as a polypeptide encoded by thereference sequence (when a polynucleotide). For example, a functionalvariant of any of the biomimetics herein described would have aspecified homology or identity and would be capable of immune modulationof DCs. Functional sequence variants include both polynucleotides andpolypeptides. Sequence identity is assessed generally using BLAST 2.0(Basic Local Alignment Search Tool), operating with the defaultparameters: Filter-On, Scoring Matrix—BLOSUM62, Word Size—3, E value—10,Gap Costs—11,1 and Alignments—50.

From the above, it will be appreciated that stradomers of the presentinvention include stradomers having: (a) only naturally occurring Fcdomains; (b) a mixture of naturally occurring Fc domains and Fc domainswith altered amino acid sequences; and (c) only Fc domains with alteredamino acid sequences. All that is required is that stradomers containingaltered amino acid sequences have at least 25%; 30%; 40%; 50%; 60%; 70%;80%; 90%; 95%; 96%; 97%; 98%; 99%; 99.5%; or 100% or even more of theability of a corresponding stradomer comprising Fc domains withnaturally-occurring sequences to bind to two or more Fcγ receptors.

The aforementioned Fcγ receptor binding sites occurring in thestradomers and stradobodies of the present invention may be altered insequence through genetic engineering to predictably derive binding siteswith altered binding capabilities and affinities relative to a nativesequence. For example, specific residues may be altered that reduce Fcdomain binding of the biomimetic compounds to FcγRII while increasingbinding to FcγRIIIa. An example of an extensive mutagenesis basedstructure-function analysis for hIgG Fcγ receptor binding sequences isRobert L. Shields, et al. High Resolution Mapping of the Binding Site onHuman IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and Design of IgG1Variants with Improved Binding to the FcγR. J. Biol. Chem., February2001; 276: 6591-6604. Similar studies have been performed on murine IgGFc (mIgG Fc). Based on the structural and primary sequence homologies ofnative IgG Fc domains across species, one of skill in the art maytranslate the extensive structure-function knowledge of hIgG Fc and mIgGFc to rational mutagenesis of all native Fcγ receptor binding sitesequences in the biomimetic compounds of the present invention to designbinding sites with particular Fcγ receptor specificities and bindingaffinities.

In addition to the amino acid sequence composition of native Fc domains,the carbohydrate content of the Fc domain is known to play an importantrole on Fc domain structure and binding interactions with FcγR. See,e.g., Robert L. Shields, et al. Lack of Fucose on Human IgG1 N-LinkedOligosaccharide Improves Binding to Human Fc RIII and Antibody-dependentCellular Toxicity. J. Biol. Chem., July 2002; 277: 26733-26740(doi:10.1074/jbc.M202069200); Ann Wright and Sherie L. Morrison. Effectof C2-Associated Carbohydrate Structure on Ig Effector Function: Studieswith Chimeric Mouse-Human IgG1 Antibodies in Glycosylation Mutants ofChinese Hamster Ovary Cells. J. Immunol., April 1998; 160: 3393-3402.Carbohydrate content may be controlled using, for example, particularprotein expression systems including particular cell lines or in vitroenzymatic modification. Thus, the present invention includes stradomersand stradobodies comprising Fc domains with the native carbohydratecontent of holo-antibody from which the domains were obtained, as wellas those biomimetic compounds have an altered carbohydrate content.

The addition to the polypeptide chain of an Fc partial domain, amultimerization region, or glycosylation changes may create aconformational change in the Fc domain permitting enhanced binding ofthe Fc domain to an Fcγ receptor. Thus, seemingly minor changes to thepolypeptide may also create a stradomer capable of binding multiple Fcγreceptors.

Partial Domains and Partial Fragments

The skilled artisan will further recognize that the Fc domains and Fcpartial domains used in the embodiments of the present invention neednot be full-length versions. That is, the present invention encompassesthe use of Fc domain monomers and Fc partial domain monomers lackingamino acids from the amino terminus, carboxy terminus or middle of theparticular Fc domain monomers and Fc partial domain monomers thatcomprise the stradomers and other biomimetics of the present invention.

For example, the binding site on human IgG immunoglobulins for Fcγreceptors has been described (e.g. Radaev, S., Sun, P., 2001.Recognition of Immunoglobulins by Fcγ Receptors. Molecular Immunology38, 1073-1083; Shields, R. L. et. al., 2001. High Resolution Mapping ofthe Binding Site on Human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn andDesign of IgG1 Variants with Improved Binding to the FcγR. J. Biol.Chem. 276 (9), 6591-6604). Based on that knowledge, one may remove aminoacids from the Fc domain of these immunoglobulins and determine theeffects on the binding interaction between the Fc domain and thereceptor. Thus, the present invention encompasses IgG Fc domains havingat least about 90% of the amino acids encompasses positions 233 through338 of the lower hinge and CH2 as defined in Radaev, S., Sun, P., 2001

Fc partial domains of IgG immunoglobulins of the present inventioninclude all or part of the hinge region, all or part of the CH2 domain,and all or part of the CH3 domain.

The IgG Fc partial domains having only a part of the hinge region, partof the CH2 domain or part of the CH3 domain are constructed from Fcpartial domain monomers. Thus, the present invention includes IgG hingeregion monomers derived from the N-terminus of the hinge region or theC-terminus of the hinge region. They can thus contain, for example, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, or 62 (up to 15 for IgG1, up to 12 for IgG2, up to 62 for IgG3, upto 12 for IgG4) amino acids of the hinge region.

The present invention also includes IgG CH2 domain monomers derived fromthe N-terminus of the CH2 domain or the C-terminus of the CH2 domain.They can thus contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, or 110 (up to 110 for IgG1 and IgG3, up to109 for IgG2 and IgG4) amino acids of the CH2 domain.

The present invention further includes IgG CH3 domain monomers derivedfrom the N-terminus of the CH3 domain or the C-terminus of the CH3domain. They can thus contain, for example, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, or 107 (up to 106 for IgG1 and IgG3, up to 107for IgG2 and IgG4) amino acids of the CH3 domain.

Fc partial domains of IgA1, IgA2 and IgD immunoglobulins of the presentinvention include all or part of the hinge region, all or part of theCH2 domain, and all or part of the CH3 domain. Moreover all or part ofthe CH1 domain of the IgA1, IgA2, or IgD immunoglobulin can be used asFc partial domains.

The IgA1, IgA2 and IgD partial domains having only a part of the hingeregion, part of the CH1 domain, part of the CH2 domain or part of theCH3 domain are constructed from Fc partial domain monomers. Thus, thepresent invention includes hinge region monomers derived from theN-terminus of the hinge region or the C-terminus of the hinge region ofIgA1, IgA2 or IgD. They can thus contain, for example, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,or 64 (up to 26 for IgA1, up to 13 for IgA2, up to 64 for IgD) aminoacids of the hinge region.

The present invention includes CH2 domain monomers derived from theN-terminus of the CH2 domain or the C-terminus of the CH2 domains ofIgA1, IgA2 or IgD. They can thus contain, for example, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, or 107 (up to 102 for IgA1, up to 96for IgA2, up to 107 for IgD) amino acids of the CH2 domain.

The present invention includes CH3 domains derived from the N-terminusof the CH3 domain or the C-terminus of the CH3 domains of IgA1, IgA2 orIgD. They can thus contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, or131 (up to 113 for IgA1, up to 131 for IgA2, up to 110 for IgD) aminoacids of the CH3 domain.

Fc partial domains of IgM and IgE immunoglobulins of the presentinvention include all or part of the hinge/CH2 domain, all or part ofthe CH3 domain, and all or part of the CH4 domain of these molecules.Moreover all or part of the CH1 domain of the IgM and IgEimmunoglobulins can be used as Fc partial domains.

The IgM and IgE partial domains having only a part of the hinge/CH2domain, part of the CH3 domain, or part of the CH4 domain areconstructed from Fc partial domain monomers. Thus, the present inventionincludes hinge/CH2 domain monomers derived from the N-terminus of thehinge/CH2 domain or the C-terminus of the hinge/CH2 domain of IgM orIgE. They can thus contain, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, or 112 (up to 112 for IgM,up to 109 for IgE) amino acids of the hinge/CH2 domain.

The present invention includes IgM and IgE CH3 domain monomers derivedfrom the N-terminus of the CH3 domain or the C-terminus of the CH3domain of IgM or IgE. They can thus contain, for example, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, or 106 (up to 106 for IgM, up to 105 forIgE) amino acids of the CH3 domain.

The present invention includes IgM and IgE CH4 domain monomers derivedfrom the N-terminus of the CH4 domain or the C-terminus of the CH4domain of IgM or IgE. They can thus contain, for example, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, or 130 (up to 130 for IgM, up to 105 for IgE) amino acids ofthe CH4 domain. However, parts of the CH4 domain of IgM or IgE thatinclude the C-terminal end of the CH4 domain will preferably be morethan 18 amino acids in length, and more preferably will be more than 30amino acids in length, and most preferably will be more than 50 aminoacids in length.

From the above, it will be appreciated that different embodiments of thepresent invention include stradomers containing: (a) full-length Fcdomains; (b) a mixture of full-length Fc domains and Fc partial domains;and (c) Fc partial domains. In each of these embodiments, the stradomersmay further comprise CH1 domains. As discussed herein, in eachembodiment of the stradomers of the present invention, the stradomershave the ability to bind two or more Fcγ receptors.

Preferred Embodiments of Stradomers and Stradomer Monomers

The following are examples of stradomer monomers of the presentinvention:

-   -   1. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3    -   2. IgG1 hinge-IgG3 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3    -   3. IgG1 hinge-IgG1 CH2-IgG3 CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3    -   4. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG3 hinge-IgG1 CH2-IgG1 CH3    -   5. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG3 CH2-IgG1 CH3    -   6. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG3 CH3    -   7. IgG1 hinge-IgG3 CH2-IgG1 CH3-IgG3 hinge-IgG1 CH2-IgG1 CH3    -   8. IgG3 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3    -   9. IgG3 hinge-IgG1 CH2-IgG1 CH3-IgG3 hinge-IgG1 CH2-IgG1 CH3    -   10. IgG3 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG3        CH3-IgG1 hinge-IgG3 CH2-IgG3 CH3    -   11. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG3 hinge-IgG3 CH2-IgG3        CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3    -   12. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG3 hinge-IgG1 hinge-IgG3        CH2-IgG3 CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3    -   13. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG3 hinge-IgG3 CH2-IgG3        CH3-IgG1 hinge-IgG2 CH2-IgG3 CH3.    -   14. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG4 hinge-IgG4 CH2-IgG4        CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3

In each of these embodiments, and the other embodiments presentedherein, it will be understood that domain linkages may be used to linkthe individual Fc partial domain monomers that make up the stradomermonomers. In one embodiment, the Fc partial domain monomers shown foreach of the stradomer monomers set forth above are human Fc partialdomain monomers.

The present invention includes stradomers comprising two or more of thestradomer monomers listed above. In preferred embodiments, the presentinvention includes serial stradomers comprising two identical stradomermonomers provided above.

As indicated above, the stradomer functionality of binding more than oneFcγ receptor can also be achieved by incorporating a J chain as a coremoiety in a core stradomer, similar to a natural IgM or IgA molecule. Innative IgA and IgM immunoglobulins the joining (J) chain is a 15 kDapeptide that joins the heavy and light chains of IgA and IgM antibodiesthrough disulfide bridges with an 18 amino acid “secretory tailpiece” ofthe Fc portions of the antibodies. Braathen, R., et al., TheCarboxyl-terminal Domains of IgA and IgM Direct Isotype-specificPolymerization and Interaction with the Polymeric ImmunoglobulinReceptor, J. Bio. Chem. 277(45), 42755-42762 (2002).

Such core stradomers may be comprised of stradomer monomers containing anaturally occurring CH4 Fc domain, preferably from IgM immunoglobulins,thereby permitting association of the stradomers comprising suchstradomer monomers to a J chain (see FIGS. 10A-10D). The following areexamples of stradomer monomers which can self-dimerize to form astradomer and then be associated with a J chain to form a core stradomercomposed of a plurality (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, fifteen, eighteen, twenty, or more) ofstradomers:

1. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3-IgM CH4(see FIGS. 10C-10D)

-   -   2. IgG1 hinge-IgG3 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3-IgM        CH4    -   3. IgG1 hinge-IgG1 CH2-IgG3 CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3-IgM        CH4 (see FIGS. 10A-10B)    -   4. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG3 hinge-IgG1 CH2-IgG1 CH3-IgM        CH4    -   5. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG3 CH2-IgG1 CH3-IgM        CH4    -   6. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3-IgM        CH4    -   7. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG3 CH3-IgM        CH4    -   8. IgG1 hinge-IgG3 CH2-IgG1 CH3-IgG3 hinge-IgG1 CH2-IgG1 CH3-IgM        CH4    -   9. IgG3 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG1 CH3-IgM        CH4    -   10. IgG3 hinge-IgG1 CH2-IgG1 CH3-IgG3 hinge-IgG1 CH2-IgG1        CH3-IgM CH4    -   11. IgG3 hinge-IgG1 CH2-IgG1 CH3-IgG1 hinge-IgG1 CH2-IgG1 hinge        IgG3 CH2-IgG3 CH3-IgM CH4

In each of these embodiments, and the other embodiments presentedherein, it will be understood that domain linkages may be used to linkthe individual Fc partial domain monomers that make up the stradomermonomers. In one embodiment, the Fc partial domain monomers shown foreach of the stradomer monomers set forth above are human Fc partialdomain monomers.

Core stradomers based on a J chain may be also be comprised of Fcfragments, Fc partial fragments and/or Fc domains that have a CH4 Fcdomain. In this example, each of the Fc fragments, Fc partial fragmentsand Fc domains having a CH4 Fc domain linked to the core moiety maycontain only one Fcγ receptor binding site but in the context of such acore stradomer, forms a biologically active biomimetic containing morethan one Fcγ receptor binding site. A skilled artisan will recognizethat the Fc partial domains from different native immunoglobulins can beused to generate the functional Fc fragments, Fc partial fragments andFc domains of such a core stradomer. The following are examples ofmonomers of Fc fragments, Fc partial fragments and Fc domains which canself-dimerize and then be associated with a J chain to form a corestradomer:

-   -   1. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgM CH4    -   2. IgG3 hinge-IgG1 CH2-IgG1 CH3-IgM CH4    -   3. IgG1 hinge-IgG3 CH2-IgG1 CH3-IgM CH4    -   4. IgG1 hinge-IgG1 CH2-IgG3 CH3-IgM CH4    -   5. IgG1 hinge-IgG3 CH2-IgG3 CH3-IgM CH4    -   6. IgG3 hinge-IgG3 CH2-IgG1 CH3-IgM CH4    -   7. IgG3 hinge-IgG3 CH2-IgG1 CH3-IgM CH4    -   8. IgG1 hinge-IgG3 CH2-IgG2 CH3-IgM CH4    -   9. IgG1 hinge-IgG3 hinge-IgG3 CH2-IgG2 CH3-IgM CH4    -   10. IgG1 hinge-IgG1 CH2-IgG1 CH3-IgE CH4-IgM CH4

In each of these embodiments, and the other embodiments presentedherein, it will be understood that domain linkages may be used to linkthe individual Fc partial domain monomers that make up the stradomermonomers. In one embodiment, the Fc partial domain monomers shown foreach of the stradomer monomers set forth above are human Fc partialdomain monomers.

It is clear from the above examples that stradomer monomers can be ofdiffering lengths and compositions to accomplish the goal, whenassociated through self-aggregation or inter-stradomer monomer linkagesto a second stradomer monomer and associated with a J chain, producing acore stradomer containing more than one Fcγ receptor binding site. Theexamples are in no way limiting and one skilled in the art willappreciate that multiple other stradomer configurations in stradomersare possible.

Fcγ Receptors

The terms “FcγR” and “Fcγ receptor” as used herein includes each memberof the Fc gamma receptor family of proteins expressed on immune cellsurfaces as described in Nimmerjahn F and Ravetch J V. Fcgammareceptors: old friends and new family members. Immunity. 2006 January;24(1):19-28, or as may later be defined. It is intended that the term“FcγR” herein described encompasses all members of the Fc gamma RI, RII,and RIII families. Fcγ receptor includes low affinity and high affinityFcγ receptors, including but not limited to FcγRI (CD64); FcγRII (CD32)and its isotypes and allotypes FcγRIIa LR, FcγRIIa HR, FcγRIIb, andFcγRIIc; FcγRIII (CD16) and its isotypes FcγRIIIa and FcγRIIIb. Askilled artisan will recognize that the present invention, whichincludes compounds that bind to FcγR, will apply to future FcγRs andassociated isotypes and allotypes that may not yet have been discovered.

It has been described that IVIG binds to and fully saturates theneonatal Fc receptor (“FcRn”) and that such competitive inhibition ofFcRn may play an important role in the biological activity of IVIG (e.g.Mechanisms of Intravenous Immunoglobulin Action in ImmuneThrombocytopenic Purpura. F. Jin, J. Balthasar. Human Immunology, 2005,Volume 66, Issue 4, Pages 403-410.) Since immunoglobulins that bindstrongly to Fcγ receptors also bind at least to some degree to FcRn, askilled artisan will recognize that stradomers which are capable ofbinding to more than one Fcγ receptor will also bind to and may fullysaturate the FcRn.

“Immunological activity of aggregated native IgG” refers to theproperties of multimerized IgG which impact the functioning of an immunesystem upon exposure of the immune system to the IgG aggregates.Specific properties of native multimerized IgG includes altered specificbinding to FcγRs, cross-linking of FcγRs on the surfaces of immunecells, or an effector functionality of multimerized IgG such as antibodydependent cell-mediated cytotoxicity (ADCC), phagocytosis (ADCP), orcomplement fixation (See, e.g., Nimmerjahn F, Ravetch J V. Theanti-inflammatory activity of IgG: the intravenous IgG paradox. J ExpMed. 2007; 204:11-15; Augener W, Friedman B, Brittinger G. Areaggregates of IgG the effective part of high-dose immunoglobulin therapyin adult idiopathic thrombocytopenic purpura (ITP)? Blut. 1985;50:249-252; Arase N, Arase H, Park S Y, Ohno H, Ra C, Saito T.Association with FcRgamma is essential for activation signal throughNKR-P1 (CD161) in natural killer (NK) cells and NK1.1+ T cells. J ExpMed. 1997; 186:1957-1963; Teeling J L, Jansen-Hendriks T, Kuijpers T W,et al. Therapeutic efficacy of intravenous immunoglobulin preparationsdepends on the immunoglobulin G dimers: studies in experimental immunethrombocytopenia. Blood. 2001; 98:1095-1099; Anderson C F, Mosser D M.Cutting edge: biasing immune responses by directing antigen tomacrophage Fc gamma receptors. J Immunol. 2002; 168:3697-3701; JefferisR, Lund J. Interaction sites on human IgG-Fc for Fc[gamma]R: currentmodels. Immunology Letters. 2002; 82:57; Banki Z, Kacani L, Mullauer B,et al. Cross-Linking of CD32 Induces Maturation of HumanMonocyte-Derived Dendritic Cells Via NF-{kappa}B Signaling Pathway. JImmunol. 2003; 170:3963-3970; Siragam V, Brinc D, Crow A R, Song S,Freedman J, Lazarus A H. Can antibodies with specificity for solubleantigens mimic the therapeutic effects of intravenous IgG in thetreatment of autoimmune disease? J Clin Invest. 2005; 115:155-160).These properties are generally evaluated by comparison to the propertiesof monomeric IgG.

“Comparable to or superior to an Fcγ receptor cross-linking or aneffector functionality of a plurality of naturally-occurring, aggregatedIgG immunoglobulins” as used herein means the stradomer generates anassay value of about 70% or more of the value achieved using 1V1G. Insome embodiments, the assay value is at least within the standard errorrange of the assay values achieved using 1V1G. In other embodiments, theassay value is 110% or higher than that of IVIG. Assays for FcγRcross-linking are well known to those of ordinary skill in the art (seee.g., Falk Nimmerjahn and Jeffrey Ravetch. Fcγ receptors as regulatorsof immune responses. Nature Reviews Immunology, advanced published online Dec. 7, 2007).

“Immune modulating activities,” “modulating immune response,”“modulating the immune system,” and “immune modulation” mean alteringimmune systems by changing the activities, capacities, and relativenumbers of one or more immune cells, including maturation of a cell typewithin its cell type or into other cell types. For example, immunemodulation of immature monocytes may lead to greater populations of moremature monocytes, dendritic cells, macrophages, or osteoclasts, all ofwhich are derived from immature monocytes. For example, immune cellreceptors may be bound by immunologically active biomimetics andactivate intracellular signaling to induce various immune cell changes,referred to separately as “activating immune modulation.” Blockadingimmune cell receptors to prevent receptor activation is also encompassedwithin “immune modulation” and may be separately referred to as“inhibitory immune modulation.”

Modulation of maturation of a monocyte refers to the differentiation ofa monocyte into a mature DC, a macrophage, or an osteoclast.Differentiation may be modulated to accelerate the rate of maturationand/or to increase the number of monocytes undergoing differentiation.Alternatively, differentiation may be reduced in terms of rate ofdifferentiation and/or number of cells undergoing differentiation.

The term “isolated” polypeptide or peptide as used herein refers to apolypeptide or a peptide which either has no naturally-occurringcounterpart or has been separated or purified from components whichnaturally accompany it, e.g., in tissues such as pancreas, liver,spleen, ovary, testis, muscle, joint tissue, neural tissue,gastrointestinal tissue, or breast tissue or tumor tissue (e.g., breastcancer tissue), or body fluids such as blood, serum, or urine.Typically, the polypeptide or peptide is considered “isolated” when itis at least 70%, by dry weight, free from the proteins and othernaturally-occurring organic molecules with which it is naturallyassociated. Preferably, a preparation of a polypeptide (or peptide) ofthe invention is at least 80%, more preferably at least 90%, and mostpreferably at least 99%, by dry weight, the polypeptide (peptide),respectively, of the invention. Since a polypeptide or peptide that ischemically synthesized is, by its nature, separated from the componentsthat naturally accompany it, the synthetic polypeptide or peptide is“isolated.”

An isolated polypeptide (or peptide) of the invention can be obtained,for example, by extraction from a natural source (e.g., from tissues orbodily fluids); by expression of a recombinant nucleic acid encoding thepolypeptide or peptide; or by chemical synthesis. A polypeptide orpeptide that is produced in a cellular system different from the sourcefrom which it naturally originates is “isolated,” because it willnecessarily be free of components which naturally accompany it. Thedegree of isolation or purity can be measured by any appropriate method,e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

Pharmaceutical Compositions

Administration of the immunologically active biomimetic compositionsdescribed herein will be via any common route, orally, parenterally, ortopically. Exemplary routes include, but are not limited to oral, nasal,buccal, rectal, vaginal, ophthalmic, subcutaneous, intramuscular,intraperitoneal, intravenous, intraarterial, intratumoral, spinal,intrathecal, intra-articular, intra-arterial, sub-arachnoid, sublingual,oral mucosal, bronchial, lymphatic, intra-uterine, subcutaneous,intratumor, integrated on an implantable device, intradural,intracortical, or dermal. Such compositions would normally beadministered as pharmaceutically acceptable compositions as describedherein. In a preferred embodiment the isolated immunologically activebiomimetic is administered intravenously.

The term “pharmaceutically acceptable carrier” as used herein includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The immunologically active biomimetic compositions of the presentinvention may be formulated in a neutral or salt form.Pharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

Sterile injectable solutions are prepared by incorporating theimmunologically active biomimetic in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Further, one embodiment is an immunologically active biomimeticcomposition suitable for oral administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimmable or edible and includes liquid,semi-solid, i.e., pastes, or solid carriers. Except insofar as anyconventional media, agent, diluent or carrier is detrimental to therecipient or to the therapeutic effectiveness of an immunologicallyactive biomimetic preparation contained therein, its use in an orallyadministrable an immunologically active biomimetic composition for usein practicing the methods of the present invention is appropriate.Examples of carriers or diluents include fats, oils, water, salinesolutions, lipids, liposomes, resins, binders, fillers and the like, orcombinations thereof. The term “oral administration” as used hereinincludes oral, buccal, enteral or intragastric administration.

In one embodiment, the composition is combined with the carrier in anyconvenient and practical manner, i.e., by solution, suspension,emulsification, admixture, encapsulation, microencapsulation, absorptionand the like. Such procedures are routine for those skilled in the art.

In a specific embodiment, the immunologically active biomimeticcomposition in powder form is combined or mixed thoroughly with asemi-solid or solid carrier. The mixing can be carried out in anyconvenient manner such as grinding. Stabilizing agents can be also addedin the mixing process in order to protect the composition from loss oftherapeutic activity through, i.e., denaturation in the stomach.Examples of stabilizers for use in an orally administrable compositioninclude buffers, antagonists to the secretion of stomach acids, aminoacids such as glycine and lysine, carbohydrates such as dextrose,mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol,mannitol, etc., proteolytic enzyme inhibitors, and the like. Morepreferably, for an orally administered composition, the stabilizer canalso include antagonists to the secretion of stomach acids.

Further, the immunologically active biomimetic composition for oraladministration which is combined with a semi-solid or solid carrier canbe further formulated into hard or soft shell gelatin capsules, tablets,or pills. More preferably, gelatin capsules, tablets, or pills areenterically coated. Enteric coatings prevent denaturation of thecomposition in the stomach or upper bowel where the pH is acidic. See,i.e., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, thebasic pH therein dissolves the coating and permits the composition to bereleased to interact with intestinal cells, e.g., Peyer's patch M cells.

In another embodiment, the immunologically active biomimetic compositionin powder form is combined or mixed thoroughly with materials thatcreate a nanoparticle encapsulating the immunologically activebiomimetic or to which the immunologically active biomimetic isattached. Each nanoparticle will have a size of less than or equal to100 microns. The nanoparticle may have mucoadhesive properties thatallow for gastrointestinal absorption of an immunologically activebiomimetic that would otherwise not be orally bioavailable.

In another embodiment, a powdered composition is combined with a liquidcarrier such as, i.e., water or a saline solution, with or without astabilizing agent.

A specific immunologically active biomimetic formulation that may beused is a solution of immunologically active biomimetic protein in ahypotonic phosphate based buffer that is free of potassium where thecomposition of the buffer is as follows: 6 mM sodium phosphate monobasicmonohydrate, 9 mM sodium phosphate dibasic heptahydrate, 50 mM sodiumchloride, pH 7.0.+/−0.1. The concentration of immunologically activebiomimetic protein in a hypotonic buffer may range from 10 microgram/mlto 100 milligram/ml. This formulation may be administered via any routeof administration, for example, but not limited to intravenousadministration.

Further, an immunologically active biomimetic composition for topicaladministration which is combined with a semi-solid carrier can befurther formulated into a cream or gel ointment. A preferred carrier forthe formation of a gel ointment is a gel polymer. Preferred polymersthat are used to manufacture a gel composition of the present inventioninclude, but are not limited to carbopol, carboxymethyl-cellulose, andpluronic polymers. Specifically, a powdered Fc multimer composition iscombined with an aqueous gel containing an polymerization agent such asCarbopol 980 at strengths between 0.5% and 5% wt/volume for applicationto the skin for treatment of disease on or beneath the skin. The term“topical administration” as used herein includes application to adermal, epidermal, subcutaneous or mucosal surface.

Upon formulation, solutions are administered in a manner compatible withthe dosage formulation and in such amount as is therapeuticallyeffective to result in an improvement or remediation of the symptoms.The formulations are easily administered in a variety of dosage formssuch as ingestible solutions, drug release capsules and the like. Somevariation in dosage can occur depending on the condition of the subjectbeing treated. The person responsible for administration can, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations meet sterility, generalsafety and purity standards as required by FDA Office of Biologicsstandards.

The route of administration will vary, naturally, with the location andnature of the disease being treated, and may include, for exampleintradermal, transdermal, parenteral, intravenous, intramuscular,intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal,intratumoral, perfusion, lavage, direct injection, and oraladministration.

The term “parenteral administration” as used herein includes any form ofadministration in which the compound is absorbed into the subjectwithout involving absorption via the intestines. Exemplary parenteraladministrations that are used in the present invention include, but arenot limited to intramuscular, intravenous, intraperitoneal,intratumoral, intraocular, or intraarticular administration.

Below are specific examples of various pharmaceutical formulationcategories and preferred routes of administration, as indicated, forspecific exemplary diseases:

Buccal or sub-lingual dissolvable tablet: angina, polyarteritis nodosa.

Intravenous: Idiopathic Thrombocytopenic Purpura, Inclusion BodyMyositis, Paraproteinemic IgM demyelinating Polyneuropathy, Necrotizingfasciitis, Pemphigus, Gangrene, Dermatomyositis, Granuloma, Lymphoma,Sepsis, Aplastic anemia, Multisystem organ failure, Multiple Myeloma andMonoclonal Gammopathy of Unknown Significance, Chronic InflammatoryDemyelinating Polyradiculoneuropathy, Inflammatory Myopathies,Thrombotic thrombocytopenic purpura, Myositis, Anemia, Neoplasia,Hemolytic anemia, Encephalitis, Myelitis, Myelopathy especiallyassociated with Human T-cell lymphotropic virus-1, Leukemia, Multiplesclerosis and optic neuritis, Asthma, Epidermal necrolysis,Lambert-Eaton myasthenic syndrome, Myasthenia gravis, Ncuropathy,Uveitis, Guillain-Barre syndrome, Graft Versus Host Disease, Stiff ManSyndrome, Parancoplastic cerebellar degeneration with anti-Yoantibodies, paraneoplastic encephalomyelitis and sensory neuropathy withanti-Hu antibodies, systemic vasculitis, Systemic Lupus Erythematosus,autoimmune diabetic neuropathy, acute idiopathic dysautonomicneuropathy, Vogt-Koyanagi-Harada Syndrome, Multifocal Motor Neuropathy,Lower Motor Neuron Syndrome associated with anti-/GM1, Demyelination,Membranoproliferative glomerulonephritis, Cardiomyopathy, Kawasaki'sdisease, Rheumatoid arthritis, and Evan's syndrome IM—ITP, CIDP, MS,dermatomyositis, mysasthenia gravis, muscular dystrophy. The term“intravenous administration” as used herein includes all techniques todeliver a compound or composition of the present invention to thesystemic circulation via an intravenous injection or infusion.

Dermal gel, lotion, cream or patch: vitiligo, Herpes zoster, acne,chelitis.

Rectal suppository, gel, or infusion: ulcerative colitis, hemorrhoidalinflammation.

Oral as pill, troche, encapsulated, or with enteric coating: Crohn'sdisease, celiac sprue, irritable bowel syndrome, inflammatory liverdisease, Barrett's esophagus.

Intra-cortical: epilepsy, Alzheimer's, multiple sclerosis, Parkinson'sDisease, Huntingdon's Disease.

Intra-abdominal infusion or implant: endometriosis.

Intra-vaginal gel or suppository: bacterial, trichomonal, or fungalvaginitis.

Medical devices: coated on coronary artery stent, prosthetic joints.

The immunologically active biomimetics described herein may beadministered in dosages from about 0.01 mg per kg to about 300 mg per kgbody weight, and especially from 0.01 mg per kg body weight to about 300mg per kg body weight, and may be administered at least once daily,weekly, biweekly or monthly. A biphasic dosage regimen may be usedwherein the first dosage phase comprises about 0.1% to about 10% of thesecond dosage phase.

Therapeutic Applications of Stradomers and Stradobodies

Based on rational design and in vitro and in vivo validations, theimmunologically active biomimetics of the present invention will serveas important biopharmaceuticals for treating autoimmune diseases and formodulating immune function in a variety of other contexts such asbioimmunotherapy for cancer and inflammatory diseases. Medicalconditions suitable for treatment with the immunologically activebiomimetics described herein include those currently routinely treatedwith hIVIG or in which hIVIG has been found to be clinically useful suchas autoimmune cytopenias, Guillain-Barre' syndrome, myasthenia gravis,anti-Factor VIII autoimmune disease, dermatomyositis, vasculitis, anduveitis (See, F. G. van der Meche, P. I. Schmitz, N. Engl. J. Med. 326,1123 (1992); P. Gajdos et al., Lancet i, 406 (1984); Y. Sultan, M. D.Kazatchkine, P. Maisonneuve, U. E. Nydegger, Lancet ii, 765 (1984); M.C. Dalakas et al., N. Engl. J. Med. 329, 1993 (1993); D. R. Jayne, M. J.Davies, C. J. Fox, C. M. Black, C. M. Lockwood, Lancet 337, 1137 (1991);P. LeHoang, N. Cassoux, F. George, N. Kullmann, M. D. Kazatchkine, Ocul.Immunol. Inflamm. 8, 49 (2000)) and those cancers or inflammatorydisease conditions in which a monoclonal antibody may be used or isalready in clinical use. Conditions included among those that may beeffectively treated by the compounds that are the subject of thisinvention include an inflammatory disease with an imbalance in cytokinenetworks, an autoimmune disorder mediated by pathogenic autoantibodiesor autoaggressive T cells, or an acute or chronic phase of a chronicrelapsing autoimmune, inflammatory, or infectious disease or process.

In addition, other medical conditions having an inflammatory componentwill benefit from treatment with immunologically active biomimetics suchas Amyotrophic Lateral Sclerosis, Huntington's Disease, Alzheimer'sDisease, Parkinson's Disease, Myocardial Infarction, Stroke, HepatitisB, Hepatitis C, Human Immunodeficiency Virus associated inflammation,adrenoleukodystrophy, and epileptic disorders especially those believedto be associated with postviral encephalitis including RasmussenSyndrome, West Syndrome, and Lennox-Gastaut Syndrome.

The general approach to therapy using the isolated immunologicallyactive biomimetics described herein is to administer to a subject havinga disease or condition, a therapeutically effective amount of theisolated immunologically active biomimetic to effect a treatment. Insome embodiments, diseases or conditions may be broadly categorized asinflammatory diseases with an imbalance in cytokine networks, anautoimmune disorder mediated by pathogenic autoantibodies orautoaggressive T cells, or an acute or chronic phase of a chronicrelapsing disease or process.

The term “treating” and “treatment” as used herein refers toadministering to a subject a therapeutically effective amount of abiomimetic of the present invention so that the subject has animprovement in a disease or condition, or a symptom of the disease orcondition. The improvement is any improvement or remediation of thedisease or condition, or symptom of the disease or condition. Theimprovement is an observable or measurable improvement, or may be animprovement in the general feeling of well-being of the subject. Thus,one of skill in the art realizes that a treatment may improve thedisease condition, but may not be a complete cure for the disease.Specifically, improvements in subjects may include one or more of:decreased inflammation; decreased inflammatory laboratory markers suchas C-reactive protein; decreased autoimmunity as evidenced by one ormore of: improvements in autoimmune markers such as autoantibodies or inplatelet count, white cell count, or red cell count, decreased rash orpurpura, decrease in weakness, numbness, or tingling, increased glucoselevels in patients with hyperglycemia, decreased joint pain,inflammation, swelling, or degradation, decrease in cramping anddiarrhea frequency and volume, decreased angina, decreased tissueinflammation, or decrease in seizure frequency; decreases in cancertumor burden, increased time to tumor progression, decreased cancerpain, increased survival or improvements in the quality of life; ordelay of progression or improvement of osteoporosis.

The term “therapeutically effective amount” as used herein refers to anamount that results in an improvement or remediation of the symptoms ofthe disease or condition.

As used herein, “prophylaxis” can mean complete prevention of thesymptoms of a disease, a delay in onset of the symptoms of a disease, ora lessening in the severity of subsequently developed disease symptoms.

The term “subject” as used herein, is taken to mean any mammaliansubject to which biomimetics of the present invention are administeredaccording to the methods described herein. In a specific embodiment, themethods of the present disclosure are employed to treat a human subject.The methods of the present disclosure may also be employed to treatnon-human primates (e.g., monkeys, baboons, and chimpanzees), mice,rats, bovines, horses, cats, dogs, pigs, rabbits, goats, deer, sheep,ferrets, gerbils, guinea pigs, hamsters, bats, birds (e.g., chickens,turkeys, and ducks), fish and reptiles to produce species-specific orchimeric stradomer molecules.

In particular, the biomimetics of the present invention may be used totreat conditions including but not limited to congestive heart failure(CHF), vasculitis, rosecea, acne, eczema, myocarditis and otherconditions of the myocardium, systemic lupus erythematosus, diabetes,spondylopathies, synovial fibroblasts, and bone marrow stroma; boneloss; Paget's disease, osteoclastoma; multiple myeloma; breast cancer;disuse osteopenia; malnutrition, periodontal disease, Gaucher's disease,Langerhans' cell histiocytosis, spinal cord injury, acute septicarthritis, osteomalacia, Cushing's syndrome, monoostotic fibrousdysplasia, polyostotic fibrous dysplasia, periodontal reconstruction,and bone fractures; sarcoidosis; osteolytic bone cancers, lung cancer,kidney cancer and rectal cancer; bone metastasis, bone pain management,and humoral malignant hypercalcemia, ankylosing spondylitisa and otherspondyloarthropathies; transplantation rejection, viral infections,hematologic neoplasisas and neoplastic-like conditions for example,Hodgkin's lymphoma; non-Hodgkin's lymphomas (Burkitt's lymphoma, smalllymphocytic lymphoma/chronic lymphocytic leukemia, mycosis fungoides,mantle cell lymphoma, follicular lymphoma, diffuse large B-celllymphoma, marginal zone lymphoma, hairy cell leukemia andlymphoplasmacytic leukemia), tumors of lymphocyte precursor cells,including B-cell acute lymphoblastic leukemia/lymphoma, and T-cell acutelymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T and NKcells, including peripheral T-cell leukemias, adult T-cellleukemia/T-cell lymphomas and large granular lymphocytic leukemia,Langerhans cell histocytosis, myeloid neoplasias such as acutemyelogenous leukemias, including AML with maturation, AML withoutdifferentiation, acute promyelocytic leukemia, acute myelomonocyticleukemia, and acute monocytic leukemias, myelodysplastic syndromes, andchronic myeloproliferative disorders, including chronic myelogenousleukemia, tumors of the central nervous system, e.g., brain tumors(glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma, andretinoblastoma), solid tumors (nasopharyngeal cancer, basal cellcarcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma,testicular cancer, uterine, vaginal or cervical cancers, ovarian cancer,primary liver cancer or endometrial cancer, tumors of the vascularsystem (angiosarcoma and hemagiopericytoma)) or other cancer.

“Cancer” herein refers to or describes the physiological condition inmammals that is typically characterized by unregulated cell growth.Examples of cancer include but are not limited to carcinoma, lymphoma,blastoma, sarcoma (including liposarcoma, osteogenic sarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, leiomyosarcoma, rhabdomyosarcoma,fibrosarcoma, myxosarcoma, chondrosarcoma), neuroendocrine tumors,mesothelioma, chordoma, synovioma, schwanoma, meningioma,adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, small cell lung carcinoma, cancer of theperitoneum, hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, testicular cancer, esophageal cancer,tumors of the biliary tract, Ewing's tumor, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, testicular tumor, lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom'smacroglobulinemia, myelodysplastic disease, heavy chain disease,neuroendocrine tumors, Schwanoma, and other carcinomas, as well as headand neck cancer.

The biomimetics of the present invention may be used to treat autoimmunediseases. The term “autoimmune disease” as used herein refers to avaried group of more than 80 diseases and conditions. In all of thesediseases and conditions, the underlying problem is that the body'simmune system attacks the body itself. Autoimmune diseases affect allmajor body systems including connective tissue, nerves, muscles, theendocrine system, skin, blood, and the respiratory and gastrointestinalsystems. Autoimmune diseases include, for example, systemic lupuserythematosus, rheumatoid arthritis, multiple sclerosis, myastheniagravis, and type 1 diabetes.

The disease or condition treatable using the compositions and methods ofthe present invention may be a hematoimmunological process, includingbut not limited to Idiopathic Thrombocytopenic Purpura,alloimmune/autoimmune thrombocytopenia, Acquired immunethrombocytopenia, Autoimmune neutropenia, Autoimmune hemolytic anemia,Parvovirus B19-associated red cell aplasia, Acquired antifactor VIIIautoimmunity, acquired von Willebrand disease, Multiple Myeloma andMonoclonal Gammopathy of Unknown Significance, Sepsis, Aplastic anemia,pure red cell aplasia, Diamond-Blackfan anemia, hemolytic disease of thenewborn, Immune-mediated neutropenia, refractoriness to platelettransfusion, neonatal, post-transfusion purpura, hemolytic uremicsyndrome, systemic Vasculitis, Thrombotic thrombocytopenic purpura, orEvan's syndrome.

The disease or condition may also be a neuroimmunological process,including but not limited to Guillain-Barré syndrome, ChronicInflammatory Demyelinating Polyradiculoneuropathy, Paraproteinemic IgMdemyelinating Polyneuropathy, Lambert-Eaton myasthenic syndrome,Myasthenia gravis, Multifocal Motor Neuropathy, Lower Motor NeuronSyndrome associated with anti-/GM1, Demyelination, Multiple Sclerosisand optic neuritis, Stiff Man Syndrome, Paraneoplastic cerebellardegeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis,sensory neuropathy with anti-Hu antibodies, epilepsy, Encephalitis,Myelitis, Myelopathy especially associated with Human T-celllymphotropic virus-1, Autoimmune Diabetic Neuropathy, or AcuteIdiopathic Dysautonomic Neuropathy.

The disease or condition may also be a Rheumatic disease process,including but not limited to Kawasaki's disease, Rheumatoid arthritis,Felty's syndrome, ANCA-positive Vasculitis, Spontaneous Polymyositis,Dermatomyositis, Antiphospholipid syndromes, Recurrent spontaneousabortions, Systemic Lupus Erythematosus, Juvenile idiopathic arthritis,Raynaud's, CREST syndrome, or Uveitis.

The disease or condition may also be a dermatoimmunological diseaseprocess, including but not limited to Toxic Epidermal Necrolysis,Gangrene, Granuloma, Autoimmune skin blistering diseases includingPemphigus vulgaris, Bullous Pemphigoid, and Pemphigus foliaccus,Vitiligo, Streptococcal toxic shock syndrome, Scleroderma, systemicsclerosis including diffuse and limited cutaneous systemic sclerosis, orAtopic dermatitis (especially steroid dependent).

The disease or condition may also be a musculoskeletal immunologicaldisease process, including but not limited to Inclusion Body Myositis,Necrotizing fasciitis, Inflammatory Myopathies, Myositis, Anti-Decorin(BJ antigen) Myopathy, Paraneoplastic Necrotic Myopathy, X-linkedVacuolated Myopathy, Penacillamine-induced Polymyositis,Atherosclerosis, Coronary Artery Disease, or Cardiomyopathy.

The disease or condition may also be a gastrointestinal immunologicaldisease process, including but not limited to pernicious anemia,autoimmune chronic active hepatitis, primary biliary cirrhosis, Celiacdisease, dermatitis herpetiformis, cryptogenic cirrhosis, Reactivearthritis, Crohn's disease, Whipple's disease, ulcerative colitis, orsclerosing cholangitis.

The disease or condition may also be Graft Versus Host Disease,Antibody-mediated rejection of the graft, Post-bone marrow transplantrejection, Post-infectious disease inflammation, Lymphoma, Leukemia,Neoplasia, Asthma, Type 1 Diabetes mellitus with anti-beta cellantibodies, Sjogren's syndrome, Mixed Connective Tissue Disease,Addison's disease, Vogt-Koyanagi-Harada Syndrome, Membranoproliferativeglomerulonephritis, Goodpasture's syndrome, Graves' disease, Hashimoto'sthyroiditis, Wegener's granulomatosis, micropolyarterits, Churg-Strausssyndrome, Polyarteritis nodosa or Multisystem organ failure.

In another embodiment, the stradomers herein described could be utilizedin a priming system wherein blood is drawn from a patient andtransiently contacted with the stradomer(s) for a period of time fromabout one half hour to about three hours prior to being introduced backinto the patient. In this form of cell therapy, the patient's owneffector cells are exposed to stradomer that is fixed on a matrix exvivo in order to modulate the effector cells through exposure of theeffector cells to stradomer. The blood including the modulated effectorcells are then infused back into the patient. Such a priming systemcould have numerous clinical and therapeutic applications.

Therapeutic Stradobody Applications in Oncology

In addition to having clinical utility for treating immunologicaldisorders, stradobodies have therapeutic use in cancer and inflammatorydisease treatment. The stradobodies may be used essentially followingknown protocols for any corresponding therapeutic antibody. Thestradobodies will generally be designed to enhance the effectdemonstrated on an effector cell by a monoclonal antibody, such as ADCCin cancer or decreased monocyte and DC maturation with decreasedcytokine release in autoimmune disease, and thereby potentiate theimmune response against the cancer relative to that which would occurusing, for example, a source monoclonal antibody for the Fab portion ofthe stradobody.

Exemplary monoclonal antibody Fab domains from which a stradobody may bedesigned includes cetuximab, rituximab, muromonab-CD3, abciximab,daclizumab, basiliximab, palivizumab, infliximab, trastuzumab,gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab,omalizumab, tositumomab, 1-131 tositumomab, efalizumab, bevacizumab,panitumumab, pertuzumab, natalizumab, etanercept, IGN101, volociximab,Anti-CD80 mAb, Anti-CD23 mAb, CAT-3888, CDP-791, eraptuzumab, MDX-010,MDX-060, MDX-070, matuzumab, CP-675,206, CAL, SGN-30, zanolimumab,adecatumumab, oregovomab, nimotuzumab, ABT-874, denosumab, AM 108, AMG714, fontolizumab, daclizumab, golimumab, CNTO 1275, ocrelizumab,HuMax-CD20, belimumab, epratuzumab, MLN1202, visilizumab, tocilizumab,ocrerlizumab, certolizumab pegol, eculizumab, pexelizumab, abciximab,ranibizimumab, mepolizumab, and TNX-355, MYO-029.

The stradomers and stradobodies, collectively immunologically activebiomimetics, disclosed herein have a number of further applications anduses.

Altering Immune Responses

The immunologically active biomimetics disclosed herein may also bereadily applied to alter immune system responses in a variety ofcontexts to affect specific changes in immune response profiles.Altering or modulating an immune response in a subject refers toincreasing, decreasing or changing the ratio or components of an immuneresponse. For example, cytokine production or secretion levels may beincreased or decreased as desired by targeting the appropriatecombination of FcRs with a stradomer designed to interact with thosereceptors. Antibody production may also be increased or decreased; theratio of two or more cytokines or immune cell receptors may be changed;or additional types of cytokines or antibodies may be caused to beproduced. The immune response may also be an effector function of animmune cell expressing a FcγR, including increased or decreasedphagocytic potential of monocyte macrophage derived cells, increased ordecreased osteoclast function, increased or decreased antigenpresentation by antigen-presenting cells (e.g. DCs), increased ordecreased NK cell function, increased or decreased B-cell function, ascompared to an immune response which is not modulated by animmunologically active biomimetic disclosed herein.

In a preferred embodiment, a subject with cancer or an autoimmune orinflammatory disease has their immune response altered comprising thestep of administering a therapeutically effective amount of animmunologically active biomimetic described herein to a subject, whereinthe therapeutically effective amount of the immunologically activebiomimetic alters the immune response in the subject. Ideally thisintervention treats the disease or condition in the subject. The alteredimmune response may be an increased or a decreased response and mayinvolve altered cytokine levels including the levels of any of IL-6,IL-10, IL-8, IL-23, IL-7, IL-4, IL-12, IL-13, IL-17, TNF-alpha andIFN-alpha. The invention is however not limited by any particularmechanism of action of the described biomimetics. The altered immuneresponse may be an altered autoantibody level in the subject. Thealtered immune response may be an altered autoaggressive T-cell level inthe subject.

For example, reducing the amount of TNF-alpha production in autoimmunediseases can have therapeutic effects. A practical application of thisis anti-TNF-alpha antibody therapy (e.g. REMICADE®) which is clinicallyproven to treat Plaque Psoriasis, Rheumatoid Arthritis, PsoriaticArthritis, Crohn's Disease, Ulcerative Colitis and AnkylosingSpondylitis. These autoimmune diseases have distinct etiologies butshare key immunological components of the disease processes related toinflammation and immune cell activity. A stradomer designed to reduceTNF-alpha production will likewise be effective in these and may otherautoimmune diseases. The altered immune response profile may also bedirect or indirect modulation to effect a reduction in antibodyproduction, for example autoantibodies targeting a subjects own tissues,or altered autoaggressive T-cell levels in the subject. For example,Multiple Sclerosis is an autoimmune disorder involving autoreactiveT-cells which may be treated by interferon beta therapy. See, e.g.,Zafranskaya M, et al., Interferon-beta therapy reduces CD4+ and CD8+T-cell reactivity in multiple sclerosis, Immunology 2007 May;121(1):29-39-Epub 2006 Dec. 18. A stradomer design to reduceautoreactive T-cell levels will likewise be effective in MultipleSclerosis and may other autoimmune diseases involving autoreactiveT-cells.

Applications in Immunological Assays

The immunologically active biomimetics disclosed herein may be used toperform immunological assays for testing the immune cell functions forwhich the immunologically active biomimetics were designed to modulate.

Signaling through low affinity Fcγ receptor pathways requires receptoraggregation and cross linking on the cell surface. These aggregation andcross linking parameters are postulated to be met through Fab binding toan antigen specific target with subsequent interaction between the Fcregion and low affinity FcγRs on the surface of responding cells. Inthis context, antibodies have the potential to evoke cellular responsesthrough two distinct pathways: 1. Fab interaction/blocking with/of anepitope specific target and 2. Fc interactions with FcRs. Despite thisknowledge, current controls for the majority of therapeutic studiesusing monoclonal antibodies employed in vivo do not adequately addressthe potential of Fc: Fcγ receptor interactions as contributors toobserved functional effects. Multiple strategies are currently employedto eliminate Fc:FcR interactions as confounding variables. For example,some studies employ Scv (single chain variable regions) or Fabfragments, which retain epitope specificity but lack the Fc region.These approaches are limited by the short half life of these reagentsand their limited potential to induce signaling. Other studies employfusion proteins composed of a receptor or ligand fused to an Fcfragment. While these types of approaches help to differentiate Fabspecific effects from those observed with receptor ligand interactions,they do not effectively control for Fc mediated effects. Evaluations ofantibody based therapeutics in animal models may also employ isotypecontrol antibodies with an irrelevant Fab binding site. The rationalefor this choice is based on presumed functional similarity betweenantibodies of the same isotype regardless of their Fab bindingspecificity or affinity. However, this use of irrelevant isotypecontrols has several fundamental flaws:

-   -   1. If the Fab fragments of these antibodies cannot bind a ligand        or antigenic epitope, it is likely that the Fc fragments will        not stimulate signaling through low affinity FcR interactions        because of the absence of Fcγ receptor cross-linking. Therefore,        observed functional differences between experimental and control        antibodies cannot be correctly attributed to Fab interaction        with an epitope specific target lacking a means to cross-link        the FcγR.    -   2. If these isotypes are produced in cells which yield different        glycoforms or different relative percentages of individual        glycoforms than the parent antibody, binding to both low and        high affinity FcRs will be altered, even if Fab affinity is        identical.

While there is no perfect control to overcome this problem, one optionis the use of isotype specific stradomers produced in the same cells asthe parent antibodies and given at a dose proportional to the expressionlevels of the epitope targeted by the experimental antibody. Forexample, the appropriate control for an epitope-specific antibodyproduced in rat would be a rat isotype-specific stradomer capable ofaggregating Fcγ receptor on the surface of effector cells.

Generally, an immune cell is exposed to an effective amount of animmunologically active biomimetic to modulate an activity of an immunecell in a known way and this immune modulation is compared to a testcompound or molecule to determine if the test compound has similarimmune modulating activity.

In another embodiment, heat aggregated stradomers, and aggregatedimmunoglobulins may be used as reagents for laboratory controls invarious immunological assays herein described and known to those ofordinary skill in the art.

Immunological assays may be in vitro assays or in vivo assays and mayinvolve human or non-human immune cells using a species-matched orspecies-unmatched immunologically active biomimetic. In one embodimentan immunological assay is performed by using an effective amount of theimmunologically active biomimetic to modulate an activity of an immunecell and comparing the modulation with a modulation of an immune cell bya test compound. The stradomer or stradobody may serve the function of apositive control reagent in assays involving the testing of othercompounds for immunological effect. The assay may compare the effect ofthe subject monoclonal antibody in comparison to the stradomer foreffector cell Fcγ receptor binding and functional response as measuredby changes in receptor expression level, cytokine release, and functionsuch as by using a Mixed Lymphocyte Reaction. In this manner, if astradomer (which lacks the Fab) generates a response which is in partsimilar to the monoclonal antibody then the monoclonal antibody's effectis, in some part, not due to specificity of its Fab but to the generaleffect of binding and cross-linking more than one Fcγ receptor on theeffector cell. The stradobody which contains both this same stradomerand the Fab from this same monoclonal antibody can further helpdistinguish the specificity of the monoclonal antibody Fab from thegeneral effect of binding and cross-linking more than one Fcγ receptoron the effector cell.

If the biological activity of a species-specific and isotype-specificantibody is replicated in part or in whole by a species-specific andisotype-specific stradomer then it is clear that Fc-Fcγ receptoractivity accounts for the portion of observed biological activityattributable to the species-specific and isotype-specific stradomer.Thus species-specific and isotype-specific stradomers are useful inassessing potential therapeutic antibodies to determine whether and towhat degree the observed biological activity is attributable either tothe Fab portion of the test antibody or to a non-specific effect of theFc portion of the molecule binding to and cross-linking more than oneFcγ receptor.

In one embodiment an isolated immunologically active biomimetic of thepresent invention comprises at least one stradomer which comprises atleast two Fc domains, or partial domains thereof, from the sameimmunoglobulin Fc class, where the immunoglobulin Fc class is selectedfrom the group consisting of IgG1, IgG2, IgG3, IgG4 and combinationsthereof. Such biomimetics are further capable of specifically binding toa first FcγRx₁, wherein x₁ is I, II, III, or IV and to a second FcγRx₂,wherein x₂ is I, II, III, or IV. These biomimetics can be furthercharacterized as having an immunological activity comprising an Fcγreceptor cross-linking or effector functionality comparable to orsuperior to an Fcγ receptor cross-linking or an effector functionalityof a plurality of naturally-occurring, aggregated IgG immunoglobulins.

In another embodiment the present invention includes an isolatedimmunologically active biomimetic that comprises at least one stradomercomprising at least two Fc domains from different immunoglobulinclasses, or partial domains thereof, wherein the biomimetic bindsspecifically to a first FcγRx₁, wherein x₁ is 1, 11, III, or IV and to asecond FcγRx₂, wherein x₂ is I, II, III, or IV. This biomimetic can befurther characterized as having an immunological activity comprising anFcγ receptor cross-linking or effector functionality comparable to orsuperior to an Fcγ receptor cross-linking or an effector functionalityof a plurality of naturally-occurring, aggregated IgG immunoglobulins toFcγRs.

In a further embodiment the present invention includes an isolatedimmunologically active biomimetic that comprises one or more stradomersthat each independently comprises three or more Fc domains, wherein thethree or more Fc domains comprise: a) a first Fc domain, wherein thefirst Fc domain comprises a Fc hinge (H) of a first immunoglobulin, b) asecond Fc domain, wherein the second Fc domain comprises a constantregion 2 (CH2) of a second immunoglobulin, wherein the second Fc domainis capable of binding specifically to a FcγRx₁, wherein x₁ is I, II,III, or IV; c) a third Fc domain, wherein the third Fc domain comprisesa constant region 3 (CH3) of a third immunoglobulin, wherein the thirdFc domain is capable of binding specifically to an FcγRx₂, wherein x₂ isI, II, III, or IV. These biomimetics may optionally comprise a fourth Fcdomain, wherein the fourth Fc domain comprises of a constant region 4(CH4) of a fourth immunoglobulin IgM. With this molecule the Fc hingemay contain at least one cysteine.

In yet another embodiment the present invention includes an isolatedimmunologically active biomimetic that comprises: a) a first Fc domainor Fc partial domain thereof, wherein the first Fc domain comprises a Fchinge (H) domain from a first immunoglobulin, wherein the Fc hingedomain comprises at least one cysteine, wherein the first Fc domaincontributes to binding specificity to a FcγRx, wherein x is I, II, III,or IV; and at least one of: i) a second Fc domain or partial domainthereof, wherein the second Fc domain comprises a constant region 2(CH2) from a second immunoglobulin which may or may not be the same asthe first immunoglobulin, wherein the second Fc domain contributes tobinding specificity to a FcγRx, wherein x is I, II, or III, IV; and,optionally, and ii) a third Fc domain or partial domain thereof, whereinthe third Fc domain comprises a constant region 3 (CH3) from a thirdimmunoglobulin, wherein the third Fc domain contributes to bindingspecificity to an FcγRx, wherein x is I, II, III, or IV; and b),optionally, a fourth Fc domain or partial domain thereof, wherein thefourth Fc domain specificity a constant region 4 (CH4) from an IgMimmunoglobulin.

In another embodiment, the isolated immunologically active biomimetic isa stradomer wherein the immunoglobulin source of the Fc domains are thesame or different and include IgA isotypes, IgG isotypes, IgD, IgE, andIgM. Another stradomer embodiment is an isolated immunologically activebiomimetic comprising a secretory signal sequence.

In one preferred embodiment the therapeutically effective amount of theisolated immunologically active biomimetics of the present invention isan amount sufficient to permit binding of the biomimetics to two or moreFcγRx, wherein x is I, II, III, or IV, on the surface of an immune cell,thereby causing the FcγRx to aggregate. The immune cell may be anyimmune effector cell such as a monocyte, a dendritic cell, a macrophage,an osteoclast, or an NK cell. The immune effector cell's maturation maybe modulated by the immunologically active biomimetic. The ratio of FcγRIIa to FcγRIIb may also become altered on the immune cell. The immunecell may be located in the plasma, bone marrow, gut, bone, lymphoidtissue, thymus, brain, a site of infection or a tumor. The functionalactivity of a macrophage, dendritic cell, osteoclast, or NK cell may bemodulated.

The therapeutically effective amount of the isolated immunologicallyactive biomimetic described herein above may be administered ex vivo toan immune cell to generate a treated immune cell followed by the step ofinfusing the treated immune cell into the subject. The treated immunecell may be a dendritic cell, macrophage, osteoclast or a monocyte.

Additional immunotherapy may be given together with any of the isolatedimmunologically active biomimetics described herein in a therapeuticallyeffective amount to the subject. The additional immunotherapy mayinclude, for example, one or more of a co-stimulatory molecule, amonoclonal antibody, a polyclonal antibody, a fusion protein, abiospecific antibody, a cytokine, an immunologically recognized antigen,a small molecule anti-cancer agent or anti-proliferative agent. Theadditional immunotherapy may be administered concurrently with orseparately from the administration of the immunologically activebiomimetic.

Cytokine (including those listed above) levels can be altered by for,example, administering one or more cytokines of interest, one or moreother cytokines that modulate the level of the one or more cytokines ofinterest, and/or antibodies (of any of the types and classes recitedherein) specific for one or more of any of the above two categories ofcytokines.

The immunologically active biomimetics described herein may be used tomodulate expression of co-stimulatory molecules from an immune cell,including a dendritic cell, a macrophage, an osteoclast, a monocyte, oran NK cell or to inhibit in these same immune cells differentiation,maturation, or cytokine secretion, including interleukin-12 (IL-12), orof increasing cytokine secretion, including interleukin-10 (IL-10), orinterleukin-6 (IL-6). A skilled artisan may also validate the efficacyof an immunologically active biomimetic by exposing an immune cell tothe immunologically active biomimetic and measuring modulation of theimmune cell function, wherein the immune cell is a dendritic cell, amacrophage, an osteoclast, or a monocyte. In one embodiment the immunecell is exposed to the immunologically active biomimetic in vitro andfurther comprising the step of determining an amount of a cell surfacereceptor or of a cytokine production, wherein a change in the amount ofthe cell surface receptor or the cytokine production indicates amodulation of the immune cell function. In another embodiment the immunecell is exposed to the immunologically active biomimetic in vivo in amodel animal for an autoimmune disease further comprising a step ofassessing a degree of improvement in the autoimmune disease.

“Capable of specifically binding to a FcγRx” as used herein refers tobinding to an FcγR, such as FcγRIII. Specific binding is generallydefined as the amount of labeled ligand which is displaceable by asubsequent excess of unlabeled ligand in a binding assay. However, thisdoes not exclude other means of assessing specific binding which arewell established in the art (e.g., Mendel C M, Mendel D B,‘Non-specific’ binding. The problem, and a solution. Biochem J. 1985 May15; 228(1):269-72). Specific binding may be measured in a variety ofways well known in the art such as surface plasmon resonance (SPR)technology (commercially available through BIACORE®) to characterizeboth association and dissociation constants of the immunologicallyactive biomimetics (Aslan K, Lakowicz J R, Geddes C. Plasmon lightscattering in biology and medicine: new sensing approaches, visions andperspectives. Current Opinion in Chemical Biology 2005, 9:538-544).

Methods Employing Fixed Fc

In order to understand the role of Fc: Fc gamma receptor (FcγR, the Fcreceptor for IgG Fc) interactions and the importance to IVIG function ofits Fc being biologically immobilized within an immunoglobulin, wecompared the effects of IVIG with both a fixed form of a recombinantIgG1 Fc fragment (rFCF) and a soluble form of a recombinant IgG1 Fcfragment (sFc) containing the hinge-CH2-CH3 domains on the function ofmonocytes during the process of differentiation from monocytes toimmature dendritic cells (iDC).

Exposure of monocytes cultured in granulocyte-macrophage colonystimulating factor (GM-CSF) and interleukin-4 (IL-4), to immobilizedrFCF and to immobilized IVIG, but not low dose soluble IVIG, enhancedCD86 expression, delayed the expression of CD11c, and suppressed theexpression of CD1a on the cells. Furthermore, these changes are likelynot secondary to non-specific protein immobilization of the rFCF onplastic, as soluble heat aggregated (sHA) IVIG, sHA rFCF or high doseIVIG (recognized to contain multimeric Fcs), induced changes similar tothose observed with immobilized rFCF.

Taken in concert, our data indicate that exposure of iDC to IVIGimmobilized on the surface of a solid, semi-solid, or gelatinoussubstrate results in a unique population of DC's (high CD86, low CD1a),capable of orchestrating immune tolerance, and that immobilizedmolecules that include the functional portion of immunoglobulin G (IgG)Fc fragments can be useful as mimetics of IVIG for the treatment oflocal and systemic inflammation, as well as a wide variety of otherpathological conditions that are, directly or indirectly, mediated bymonocyte derived cells (MDC) such as iDC. Moreover, immobilizing thefunctional portion of IgG Fc on devices, described herein as “coatingdevices”, that are implanted into the bodies or attached to the bodiesof animals (e.g., human patients) with molecules containing thefunctional portion of IgG Fc fragment can lessen, if not prevent,inflammatory responses to such devices.

The invention provides a method of inhibiting the activity of amonocyte-derived cell (MDC). The method includes contacting the cellwith a composition comprising a substrate with an Fc reagent boundthereto. The contacting can be in vitro, in vivo, or ex vivo.Alternatively, the cell can be in an animal. The animal can be one thathas, or is at risk of developing, a monocyte derived cell mediatedcondition (MDCMC). The MDC can be, for example, a dendritic cell, amacrophage, a monocyte, or an osteoclast.

The invention also provides a method of treatment or prophylaxis. Themethod that includes administering to an animal a composition containinga substrate having an Fc reagent bound to it, the animal being one thathas or is at risk of developing a MDCMC.

As used herein, the term “monocyte-derived cell mediated condition(MDCMC)” refers to a pathologic condition that is directly orindirectly, partially or wholly, due to the activity of, or factorsproduced by, monocyte-derived cells. Monocyte-derived cells include, butare not limited to, monocytes, macrophages, interdigitating dendriticcells (generally referred to herein as “dendritic cells” comprisingdendritic-like cells and follicular dendritic-like cells) (mature andimmature), osteoclasts, microglia-like cells, monocyte derivedinsulin-producing islet-like cells, monocyte-derived immature mast cellsand monocyte-derived microparticles.

With respect to methods using fixed Fc, the term “Fc reagent” refers toany molecule, or molecular complex, that includes one or more (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, or more) functional portions ofan immunoglobulin Ig (IgG) Fc fragment. The Fc fragment of IgG consistsof the C-terminal portions of the two IgG heavy chains of an IgGmolecule linked together and consists of the hinge regions, the CH2domains, and the CH3 domains of both heavy chains linked together. The“functional portion of the IgG Fc fragment” consists of the hingeregions, the CH2 domains, and optionally, all or some (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, or 49) of the first 50 (from the N-terminus)amino acids of the CH3 domains, of both heavy chains linked together. Inhumans, (a) the IgG1 hinge region contains 15 amino acids, the CH2domain contains 110 amino acids, and the CH3 domain contains 106 aminoacids; (b) the IgG2 hinge region contains 12 amino acids, the CH2 domaincontains 109 amino acids, and the CH3 domain contains 107 amino acids;(c) the IgG3 hinge region contains 62 amino acids, the CH2 domaincontains 104 amino acids, and the CH3 domain contains 106 amino acids;and (d) the IgG4 hinge region contains 12 amino acids, the CH2 domaincontains 109 amino acids, and the CH3 domain contains 107 amino acids.

As in wild-type IgG molecules, in the above-described Fc reagents thetwo polypeptide chains derived from IgG heavy chains are generally, butnot necessarily, identical. Thus, an Fc reagent can be, withoutlimitation, a whole IgG molecule, a whole IgG molecule linked to anon-immunoglobulin derived polypeptide, an IgG Fc fragment, an IgG Fcfragment linked to a non-immunoglobulin derived polypeptide, afunctional portion of an IgG Fc fragment, a functional portion of an IgGFc fragment linked to a non-immunoglobulin derived polypeptide ormultimers (e.g., dimers, trimers, tetramers, pentamers, hexamers,heptamers, octamers, nonamers, or decamers) of any of these. Fc reagentscan also be the above-described stradomers and stradobodies providedthat they fall within the definition of a Fc reagent above.

In the fixed Fc, immunoglobulin heavy chain components of the Fcreagents can have wild-type amino acid sequences or they can bewild-type amino acid sequences but with not more than 20 (e.g., not morethan: 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,or 1) amino acid substitutions. Such substitutions are preferably, butnot necessarily, conservative substitutions. Conservative changestypically include changes within the following groups: glycine andalanine; valine, isoleucine, and leucine; aspartic acid and glutamicacid; asparagine, glutamine, serine and threonine; lysine, histidine andarginine; and phenylalanine and tyrosine.

An “Fc reagent” of the invention has least 25% (e.g., at least: 30%;40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; or 100% or evenmore) of the ability of the IgG molecule from which the IgG heavy chaincomponents of the Fc reagent were derived (the reference IgG molecule)to bind to an Fc receptor of interest. Where an “Fc reagent” has heavychain components derived from more than one type of IgG molecule, thereference IgG molecule is the one that binds with the greatest avidityto the relevant Fc receptor of interest.

As used herein “fixed Fc” refers to an Fc reagent that is bound to a“substrate” as defined below. The terms “fixed Fc,” “bound Fc” and“stabilized Fc” are synonymous terms. Fixed Fc is comprised of thefunctional portion of Fc (including but not limited to any polypeptidethat includes the functional portion of Fc) attached to a substrate.Fixed Fc includes, for example, direct binding as well as indirectbinding through polymers of Fc to substrate; incorporation of the fullIgG Fc in isolation; incorporation of only the functional domains of IgGFc; or incorporation of the full IgG Fc or functional domains of IgG Fcas part of a larger polypeptide such as an antibody, a stradomer, or astradobody.

As applied to fixed Fc, the term “substrate” refers to a solid,semi-solid, or gelatinous object. The substrate can be implanted in, orattached (or adhered) to the surface of, the body of an animal. Thesubstrates can include, for example, liquid or gaseous components but atleast a portion of the substrate is solid, semi-solid, or gelatinous.Thus, a substrate can be a substance that is substantially insoluble inan aqueous solvent but soluble in a non-aqueous solvent. Such substancesinclude lipids (e.g., phospholipids), fatty acids, and otherfat-soluble, aqueous solvent-insoluble compounds. From this, it will beclear that substrates include liposomes. The substrate may be porous ornon-porous. In certain embodiments, the substrate is inert to thesurface and/or body to which it is implanted, attached, or adhered.

The substrate can contain or be made of a synthetic polymer, e.g.,nylon, teflon, dacron, polyvinyl chloride, PEU (poly(ester urethane)),PTFE (polytetrafluoroethylene), PMMA (methyl methacrylate) PEEK,thermoplastic elastomers, radiopaque polymers, polyethersulfone,silicons, polycarbonates, polyurethanes, polyisobutylene and itscopolymers, polyesters, polyolefins, polyisobutylene,ethylene-alphaolefin copolymers, acrylic polymers and copolymers, vinylhalide polymers and copolymers such as polyvinyl chloride, polyvinylethers, polyvinyl methyl ether, polyvinylidene halides, polyvinylidenefluoride, polyvinylidene chloride, polyacrylonitrile, polyvinyl ketones,polyvinyl aromatics, polystyrene, polyvinyl esters, polyvinyl acetate,copolymers of vinyl monomers, copolymers of vinyl monomers and olefins,ethylene-methyl methacrylate copolymers, acrylonitrile-styrenecopolymers, ABS resins, ethylene-vinyl acetate copolymers, polyamides,Nylon 66, polycaprolactone, alkyd resins, polyoxyethylenes, polyimides,polyethers, epoxy resins, rayon-triacetate, cellulose, celluloseacetate, cellulose butyrate, cellulose acetate butyrate, cellophane,cellulose nitrate, cellulose propionate, cellulose ethers, carboxymethylcellulose, collagens, chitins, polylactic acid, polyglycolic acid,polylactic acid-polyethylene oxide copolymers, polysiloxanes,substituted polysiloxanes, ethylene vinyl acetate copolymers, polyolefinelastomers, and EPDM rubbers, and combinations thereof.

The substrate can also contain or be made of a metal or a metal alloy,e.g., stainless steel, platinum, iridium, titanium, tantalum,nickel-titanium alloy, or cobalt-chromium alloy. Moreover, the substratecan include or be an animal tissue or an animal tissue product, e.g., atissue or organ graft. The animal tissue can be, for example, bone(e.g., osteogenic bone) or cartilage. Furthermore, the substrate cancontain a protein, e.g., collagen or keratin. The substrate can also beor contain a tissue matrix, e.g., an acellular tissue matrix.Particulate and non-particulate acellular matrices are described indetail in, for example, U.S. Pat. Nos. 5,336,616 and 6,933,326, thedisclosures of which are incorporated herein by reference in theirentirety. The substrate can also be or include an animal cell (e.g.,tissue repair cells such as fibroblasts; mesenchymal stem cells) and itcan be, for example, a hair transplant plug. The substrate can containor be a polysaccharide, e.g., agarose. It can also contain or be a salt,preferably a relatively insoluble salt, e.g., calcium sulfate. Thesubstrate can be a gel or cream. Moreover, it can contain silicon orsilastic. Substrates can also contain a natural fiber, e.g., silk,cotton, or wool.

In addition, the substrate can be an implantable medical device. It canbe, for example, a stent (e.g., a vascular stent such as a coronaryartery stent; an airway stent such as an endotracheal or nasal stent; agastrointestinal stent such a biliary or pancreatic stent; or a urinarystent such as a ureteral stent) or a surgical suture (e.g., a braidsilk, chromic gut, nylon, plastic, or metal suture) or a surgical clip(e.g., an aneurism clip). The substrate can be, for example, anartificial hip, an artificial hip joint, an artificial knee, anartificial knee joint, an artificial shoulder, an artificial shoulderjoint, an artificial finger or toe joint, a bone plate, a bone dowel, abone non-union implant, an intervertebral disk implant, bone cement, ora bone cement spacer. It can also be an arterial-venous shunt, animplantable wire, a pacemaker, an artificial heart, a heart assistdevice, a cochlear implant, an implantable defibrillator, a spinal cordstimulator, a central nervous system stimulator, or a peripheral nerveimplant. Other substrates are dental prostheses or dental crowns.

In other embodiments, the substrate can be a large vessel embolicfiltering device or cage, a percutaneous device, a dermal or sub-mucosalpatch, or an implantable drug delivery device. The substrate can also bea large blood vessel graft, wherein the blood vessel is, for example, acarotid artery, a femoral artery, or an aorta. Moreover, the substratecan be a sub-dermal implant, a corneal implant, an intraocular lens, ora contact lens.

The substrate can be in the form of a sheet, a bead, a mesh, a powderparticle, a thread, a bead, or a fiber. It can also include or be asolid, a semi-solid or a gelatinous substance.

Polymers useful in the invention are preferably those that arebiostable, biocompatible, particularly during insertion or implantationof the device into the body, and avoid irritation to body tissue.

Fc reagents can be coated (i.e., fixed or stabilized) onto substrates inany of a variety of manners. For example, they can be coated directly onthe surface of substrates where they remain attached by, for example,hydrophobic interactions. Below are described a few other methodologies((a)-(e)) involving the use of polymers:

(a) The Fc reagent is mixed with a miscible polymer blend which is thenlayered on to the surface of the implantable synthetic material, therebystabilizing the Fc reagent. Monomers routinely used in the art to makepolymer blends include PLMA [poly(lauryl methacrylate)]; PEG[polyethylene glycol], PEO [polyethylene oxide]; the alkylfunctionalized methacrylate polymers PMMA, PEMA, PPMA, and PBMA;itaconates; fumarates; and styrenics.

(b) A polymeric undercoat layer or a nanometer dimension film is adheredto the substrate surface and then the Fc reagent is adhered to thepolymeric undercoat layer or nanometer dimension film, therebystabilizing the F reagent.

(c) A thin film of a polymer monomer is applied to the implantablesubstrate surface and the monomer is then caused to polymerize Suchmonomers include, for example, Methane, Tetrafluorethylene, Benzene,Methanol, Ethylene oxide, Tetraglyme, Acrylic acid, Allylamine,Hydroxyethyl methacrylate, N-vinyl pyrrolidone, and mercaptoethanol. TheFc reagent is then attached to the resulting monomer.

(d) The substrate is coated with a protein such as protein A or albuminwhich attaches to the Fc reagent, thereby stabilizing Fc to the surfaceof the substrate.

(e) The Fc reagent can be tagged with a chain of hydrophobic amino acidsthat bind to implantable synthetic materials and cause the stabilized Fcto orient uniformly.

The methods of the invention can be applied to any animal species andthe IgG molecules from which the IgG-derived portions of Fc reagents aremade can be from any animal species. Naturally, relevant animal speciesare those in which IgG or IgG-like molecules occur. Generally thespecies to which the methods are applied and the species from which theIgG-derived portions of the Fc reagents used in the methods are thesame. However, they are not necessarily the same. Relevant animalspecies are preferably mammals and these include, without limitation,humans, non-human primates (e.g., monkeys, baboons, and chimpanzees),horses, bovine animals (e.g., bulls, cows, or oxen), pigs, goats, sheep,dogs, cats, rabbits, gerbils, hamsters, rats, and mice. Non-mammalianspecies include, for example, birds (e.g., chickens, turkeys, and ducks)and fish.

The terms “treating”, “treatment”, and “prophylaxis” have the samemeaning using fixed Fc as described above for stradomers andstradobodies.

Where the fixed Fc are implantable devices coated with Fc reagents, theycan be implanted in, attached to, or adhered to relevant internal organsor tissue or body surfaces of relevant subjects using methods well knownin the art. Where they are formulated as, for example, suspensions,powders, they can be formulated and administered as described above forstradomers and stradobodies.

The fixed Fc reagents of the present invention may be used to treat orprevent conditions including but not limited to cancer, congestive heartfailure (CHF), vasculitis, rosecea, acne, eczema, myocarditis and otherconditions of the myocardium, systemic lupus erythematosus, diabetes,spondylopathies, synovial fibroblasts, and bone marrow stroma; boneloss; Paget's disease, hypertrophic bone formation; disuse osteopenia;malnutrition, periodontal disease, Gaucher's disease, Langerhans' cellhistiocytosis, spinal cord injury, acute septic arthritis, osteomalacia,Cushing's syndrome, monoostotic fibrous dysplasia, polyostotic fibrousdysplasia, periodontal reconstruction, and bone fractures, bone painmanagement, and humoral malignant hypercalcemia, ankylosing spondylitisand other spondyloarthropathies; transplantation rejection, and viralinfections.

All autoimmune diseases may be in part or in whole an MDCMD. The term“autoimmune disease” as used herein refers to a varied group of morethan 80 chronic illnesses. In all of these diseases, the underlyingproblem is that the body's immune system attacks the body itself.Autoimmune diseases affect all major body systems including connectivetissue, nerves, muscles, the endocrine system, skin, blood, and therespiratory and gastrointestinal systems.

The autoimmune disease or condition may be a hematoimmunologicalprocess, including but not limited to Idiopathic ThrombocytopenicPurpura, alloimmune/autoimmune thrombocytopenia, Acquired immunethrombocytopenia, Autoimmune neutropenia, Autoimmune hemolytic anemia,Parvovirus B19-associated red cell aplasia, Acquired antifactor VIIIautoimmunity, acquired von Willebrand disease, Multiple Myeloma andMonoclonal Gammopathy of Unknown Significance, Sepsis, Aplastic anemia,pure red cell aplasia, Diamond-Blackfan anemia, hemolytic disease of thenewborn, Immune-mediated neutropenia, refractoriness to platelettransfusion, neonatal, post-transfusion purpura, hemolytic uremicsyndrome, systemic Vasculitis, Thrombotic thrombocytopenic purpura, orEvan's syndrome.

The autoimmune disease or condition may be a neuroimmunological process,including but not limited to Guillain-Barré syndrome, ChronicInflammatory Demyelinating Polyradiculoneuropathy, Paraproteinemic IgMdemyelinating Polyneuropathy, Lambert-Eaton myasthenic syndrome,Myasthenia gravis, Multifocal Motor Neuropathy, Lower Motor NeuronSyndrome associated with anti-/GM1, Demyelination, Multiple Sclerosisand optic neuritis, Stiff Man Syndrome, Paraneoplastic cerebellardegeneration with anti-Yo antibodies, paraneoplastic encephalomyelitis,sensory neuropathy with anti-Hu antibodies, epilepsy, Encephalitis,Myelitis, Myelopathy especially associated with Human T-celllymphotropic virus-1, Autoimmune Diabetic Neuropathy, or AcuteIdiopathic Dysautonomic Neuropathy.

The autoimmune disease or condition may be a Rheumatic disease process,including but not limited to Kawasaki's disease, Rheumatoid arthritis,Felty's syndrome, ANCA-positive Vasculitis, Spontaneous Polymyositis,Dermatomyositis, Antiphospholipid syndromes, Recurrent spontaneousabortions, Systemic Lupus Erythematosus, Juvenile idiopathic arthritis,Raynaud's, CREST syndrome, or Uveitis.

The autoimmune disease or condition may be a dermatoimmunologicaldisease process, including but not limited to Toxic EpidermalNecrolysis, Gangrene, Granuloma, Autoimmune skin blistering diseasesincluding Pemphigus vulgaris, Bullous Pemphigoid, and Pemphigusfoliaceus, Vitiligo, Streptococcal toxic shock syndrome, Scleroderma,systemic sclerosis including diffuse and limited cutaneous systemicsclerosis, or Atopic dermatitis (especially steroid dependent).

The autoimmune disease or condition may be a musculoskeletalimmunological disease process, including but not limited to InclusionBody Myositis, Necrotizing fasciitis, Inflammatory Myopathies, Myositis,Anti-Decorin (BJ antigen) Myopathy, Paraneoplastic Necrotic Myopathy,X-linked Vacuolated Myopathy, Penacillamine-induced Polymyositis,Atherosclerosis, Coronary Artery Disease, or Cardiomyopathy.

The autoimmune disease or condition may be a gastrointestinalimmunological disease process, including but not limited to perniciousanemia, autoimmune chronic active hepatitis, primary biliary cirrhosis,Celiac disease, dermatitis herpetiformis, cryptogenic cirrhosis,Reactive arthritis, Crohn's disease, Whipple's disease, ulcerativecolitis, or sclerosing cholangitis.

The autoimmune disease or condition may be Graft Versus Host Disease,Antibody-mediated rejection of the graft, Post-bone marrow transplantrejection, Post-infectious disease inflammation, Lymphoma, Leukemia,Neoplasia, Asthma, Type 1 Diabetes mellitus with anti-beta cellantibodies, Sjogren's syndrome, Mixed Connective Tissue Disease,Addison's disease, Vogt-Koyanagi-Harada Syndrome, Membranoproliferativeglomerulonephritis, Goodpasture's syndrome, Graves' disease, Hashimoto'sthyroiditis, Wegener's granulomatosis, micropolyarterits, Churg-Strausssyndrome, Polyarteritis nodosa or Multisystem organ failure.

“Cancer” herein refers to or describes the physiological condition inmammals that is typically characterized by unregulated cell growth.Examples of cancer include but are not limited to carcinoma, lymphoma,blastoma, sarcoma (including liposarcoma, osteogenic sarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, leiomyosarcoma, rhabdomyosarcoma,fibrosarcoma, myxosarcoma, chondrosarcoma), osteoclastoma,neuroendocrine tumors, mesothelioma, chordoma, synovioma, schwanoma,meningioma, adenocarcinoma, melanoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, smallcell lung carcinoma, cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer including gastrointestinal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,testicular cancer, esophageal cancer, tumors of the biliary tract,Ewing's tumor, basal cell carcinoma, adenocarcinoma, sweat glandcarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testiculartumor, lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, multiplemyeloma, Waldenstrom's macroglobulinemia, myelodysplastic disease, heavychain disease, neuroendocrine tumors, Schwanoma, and other carcinomas,head and neck cancer, myeloid neoplasias such as acute myelogenousleukemias, including AML with maturation, AML without differentiation,acute promyelocytic leukemia, acute myelomonocytic leukemia, and acutemonocytic leukemias, myelodysplastic syndromes, and chronicmyeloproliferative disorders, including chronic myelogenous leukemia,tumors of the central nervous system, e.g., brain tumors (glioma,neuroblastoma, astrocytoma, medulloblastoma, ependymoma, andretinoblastoma), solid tumors (nasopharyngeal cancer, basal cellcarcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma,testicular cancer, uterine, vaginal or cervical cancers, ovarian cancer,primary liver cancer or endometrial cancer, tumors of the vascularsystem (angiosarcoma and hemagiopericytoma), hematologic neoplasias andneoplastic-like conditions for example, Hodgkin's lymphoma;non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocyticlymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle celllymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginalzone lymphoma, hairy cell leukemia and lymphoplasmacytic leukemia),tumors of lymphocyte precursor cells, including B-cell acutelymphoblastic leukemia/lymphoma, and T-cell acute lymphoblasticleukemia/lymphoma, thymoma, tumors of the mature T and NK cells,including peripheral T-cell leukemias, adult T-cell leukemia/T-celllymphomas and large granular lymphocytic leukemia, ostcolytic bonecancers, and bone metastasis.

As used herein, a subject “at risk of developing a monocyte-derived cellmediated disease (MDCMD)” is a subject that has a predisposition todevelop the MDCMD, i.e., a genetic predisposition to develop the MDCMDor has been exposed to conditions that can result in MDCMD. A subject“suspected of having a MDCMD” is one having one or more symptoms of aMDCMD. From the above it will be clear that neither subjects “at risk ofdeveloping a MDCMD” nor subjects “suspected of having a MDCMD” are allindividuals within a species of interest.

In any of the above methods, the MDCMC can be one caused by thesubstrate and the Fc reagent serves to prevent or ameliorate the MDCMC.

Example 1 Construct Design of Immunologically Active Biomimetics

A sequence encoding a Fc fragment monomer from human IgG₁ (SEQ ID NO: 1)has been cloned into an expression vector (pCDNA 3.1D/V5 His TOPOInvitrogen) comprising selected restriction enzyme cleavage sites, anIgK signal (further defined below) and epitope tags to create the IgG1monomer sequence {RestEnzSites-IgK signal-RestEnzSites-IgG1(Hinge-CH2-CH3)-RestEnzSites-epitope tags (V5 and His)-STOP}, shown inFIG. 17 (SEQ ID NO:19). The construct was transfected into CHO cells(CHO-002) for protein production. Additionally, we have designed severalstradomer constructs with the general structures:

a) {RestEnzSites-IgK signal-RestEnzSites-IgG1(Hinge-CH2-CH3)-XbaIsite-IgG1(Hinge-CH2-CH3)-STOP} (SEQ ID NO: 21) (see also FIG. 4A andFIG. 18);

b) {RestEnzSites-IgK signal-RestEnzSites-IgG1(Hinge-CH2-CH3)-XbaIsite-IgG1(Hinge-CH2-CH3)-RestEnzSites-epitope tags (V5 and His)-STOP}(SEQ ID NO: 23) (see also FIG. 19);

c) {RestEnzSites-IgK signal-EcoRVSite-IgG3(Hinge-CH2-CH3)-IgG1(Hinge-CH2-CH3)-RestEnzSites-epitopetags(V5 and His)-STOP} (SEQ ID NO.: 25) (see also FIG. 21); and

d) {RestEnzSites-IgK signal-EcoRVSite-IgE(CH2)-IgG1(Hinge-CH2-CH3)-IgG1(Hinge-CH2)-IgE(CH4)-STOP} (SEQ IDNO.: 27) (see also FIG. 22).

The IgG₁ stradomer construct a) (SEQ ID NO: 21; FIG. 18) was engineeredusing PCR. Primers complementary to the hinge sequence (at the 5′ end)of IgG₁ (SEQ ID NO: 29) and to the C terminus of the IgG₁ (at the 3′end) (SEQ ID NO: 30) were used to amplify the IgG₁ Hinge-Fc region.Restriction sites were added to the primers to permit in-frame cloningof the second Fc domain in series with the first, which was cloned intoa pcDNA cloning vector (pCDNA 3.1 D/V5 His TOPO, Invitrogen). A stopcodon was added before the restriction site of the C terminal primer toprevent read through of flanking sequences for this construct.

The stradomer construct b) (SEQ ID NO: 23; FIG. 19), was similarly madeand contained the IgG₁ Fc-IgG₁ Fc as described above but also containedtwo epitope tags added to the C terminus of the construct. These epitopetags are used for identification or purification of the protein. In thissecond construct the two epitope tags, V5 and His tag, are present inframe prior to the stop codon.

Proteins that are normally secreted routinely contain a hydrophobicsignal sequence at the N terminus of the protein. For the stradomerconstructs, we used the IgK signal sequence METDTLLLWVLLLWVPGSTG (SEQ IDNO:35) which is removed from the protein as it is secreted by mammaliancells such as Chinese Hamster Ovary cells. The predicted cleavage sitewas based on algorithms for signal site cleavage prediction (SignalP3.0).

Additional stradomer constructs, similar to a) and b) above were madethat contained the IgG₁ Fc-IgG₁ Fc structure as described above (withand without the epitope tag) but using the IgG₃ Hinge domain in theconstruct: IgG₁ Fc-IgG₃ hinge-IgG₁ (CH2-CH3).

Example 2 Design and Testing of Immunologically Active Biomimetics

Coated IVIG and Coated Fc Stimulate Similar Phenotypic Changes

IVIG and Fc when coated onto the walls and floor of the wells of asterile plate stimulate nearly identical changes in CD1a and CD86 levelson immature DC and delay the up regulation of CD11c. Because of therecognized critical role of DC in ITP, these data provide a rationalmodel for evaluating the function of IVIG mimetics such as stradomers.We also conclude that the fact that the phenotypic changes induced byIVIG are completely recapitulated by recombinant Fc suggest that theeffects of IVIG on DC are highly likely to be Fc mediated.

Stradomer Generation

We have constructed stradomers of four different classes to mimic theeffects of IVIG on immature DC. The serial stradomers, cluster stradomerunits comprising cluster stradomers, core stradomer units comprisingcore stradomers, and Fc fragment stradomers shown below in Table 3 wereeach produced except where noted. To obtain the appropriate sequencesfor each of the human constructs shown below, cDNA was synthesized fromtotal RNA extracted from human PBMC. To obtain those sequences fromother species RNA was purified from tissue of those species. Randompriming was used to produce the cDNA. The cDNA was used to amplify thedesired fragments using PCR to synthesize, clone and subsequentlycharacterize by sequence analysis the DNA fragments. The finalconstructs were produced by either sewing by overlap extension with PCR(Horton R M, Hunt H D, Ho S N, Pullen, J K and Pease L R. Engineeringhybrid genes without the use of restriction enzymes: gene splicing byoverlap extension. Gene 77:61-68, 1989) or utilized existing compatiblerestriction sites to fuse the appropriate fragments.

For example, in the cloning of G-007, the IgECH4 domain was directlyfused to the CH2 domain of IgG1 at the 3′ end of the protein. This wasaccomplished by making primers that contain the overlapping sequencesfor IgG1CH2 (C terminus) with the N terminal amino acids for IgECH4. Inone case, a hybrid primer was used to amplify 5′ with IgG1 sequences andthe complementary primer was used to amplify with a 3′ primer from the Cterminus of the IgECH4. Products from these two reactions were mixed andthe flanking primers were used to amplify the fusion protein. Sequenceanalysis confirmed the construct.

In many cases, restriction sites were utilized that were convenientlypresent at the ends of the molecules to be joined. When restrictionsites are in fusion there are detectable remnant restriction sequencesat the ends of the linked sequences. This approach was used for most ofthe constructs shown below in Table 3. The amino acid sequences of thestradomers shown in Table 3 are provided in FIG. 24. Some of thesequence are shown with His-tags, known in the art to be useful inpurifying proteins.

TABLE 3 N-term CH2 CH3 CH2 CH3 H CH2 CH3 Serial Stradomers Hin Hin G-003IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 G-004 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 G-007IgECh2 IgG1 IgG1 IgG1 IgG1 IgG1 IgCh4 G-011 IgECh2 IgG1 IgG1 IgG1 IgG1IgG1 IgG1 G-012 IgECh2 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgCh4 G-012 IgECh2IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgCh4 G-014 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1IgG1 IgG1 IgG1 G-016 IgG3 IgG3 IgG3 IgG1 IgG1 IgG1 (x-b) RestEnz G-017linker IgG1 IgG IgG1x-b IgG1 IgG1 IgG1 G-023 IgG1 IgG1 IgG1 IgG1 IgG1IgG1 Ig3H 32/62 G-024 IgG3 IgG3 IgG3 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 G-025107aa IgG1 IgG1 IgG1 G-026 107aa IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 FcFragment Stradomer and Core Stradomer Components G-002 IgG1 IgG1 IgG1G-022 IgG1 IgG1 IgG1 IgG3Hing Cluster Stradomer Units G-008 IgG1 IgG1IgG1 IgG1 IgG1 IgG1 IgM CH3-CH4-TP G-009 IgG1 IgG1 IgG1 IgMCH3-CH4-TPG-010 IgECh2 IgG1 IgG1 IgG1 G-018 IgG2 IgG2 IgG2 IgG1 IgG1 IgG1 G-019IgG2Hing IgG1 IgG1 IgG G-020 IgG2Hing IgG1 IgG1 IgG1 IgG1 IgG1 IgG1G-021 IgG2Hing IgG1 IgG1 IgG1 G-027 IgECh2-IgECh2 IgG1 IgG1 IgG1 G-028ILZ IgG1 IgG1 IgG1 G-029 ILZ-IgECh2 IgG1 IgG1 IgG1 G-030 ILZ IgG2 IgG2IgG2 IgG1 IgG1 IgG1 G-031 ILZ-IgG2hing IgG1 IgG1 IgG1 G-032 ILZ-ILZ IgG1IgG1 IgG1 G-033 IgG2Hing-IgECh2 IgG1 IgG1 IgG G-034 IgG2hing-ILZ IgG2IgG2 IgG2 IgG1 IgG1 IgG1 G-035 IgG2hing -IgG2hing IgG1 IgG1 IgG1 G-036IgG2hing-ILZ IgG1 IgG1 IgG1 To Be Made H H 401 IgG1 IgG1 IgG1 IgG3 IgG3IgG3 402 IgG3 IgG1 IgG1 IgG3 IgG1 IgG1 403 IgG1 IgG3 IgG1 IgG1 IgG3 IgG1404 IgG1 IgG1 IgG3 IgG1 IgG1 IgG3 405 IgG3 IgG3 IgG1 IgG3 IgG3 IgG1 406IgG1 IgG1 none IgG3 IgG3 none 407 IgG3 IgG3 IgG3 IgG3 IgG3 IgG3 IgG3IgG3 IgG3 408 IgG1 IgG1 IgG1 IgG3 IgG3 IgG3 IgG1 IgG1 IgG1 409 IgG3 IgG3IgG3 IgG1 IgG1 IgG1 IgG3 IgG3 IgG3 410 IgG3 IgG1 IgG1 IgG3 IgG1 IgG1IgG3 IgG1 IgG1 411 IgG3 IgG3 IgG1 IgG3 IgG3 IgG1 IgG3 IgG3 IgG1 412 IgG1IgG1 IgG4CH4 IgG3 IgG3 IgG4CH4 IgG1 IgG1 IgGCH4 413 IgG1 IgG1 IgG3 IgG3IgG1 IgG1 414 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 IgG1 415 IgG3 IgG3IgG3 IgG3 IgG3 IgG3 IgG3 IgG3 IgG3Stradomer Protein Expression

For protein expression of the stradomers, plasmid DNA encoding thestradomers described above were transfected into CHO suspension cells(Freestyleim MAX CHO expression system, Invitrogen CA). Followingprotein expression the expressed stradomers were purified from theculture media by affinity column chromatography using protein A orprotein G affinity columns. Purified stradomers were analyzed bySDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)under reducing conditions followed by Coomasie Blue staining to confirmthe presence of monomeric protein bands of expected size as exemplified:G-002: approximately 35 KD band, G-004 approximately 70 KD band, G-010:approximately 45 KD band, G-011: approximately 80 KD band, G-012:approximately 85 KD band, G-018 approximately 70 KD band, G-019:approximately 35 KD, G-028 approximately 37 KD band. Plasmid DNAencoding the stradomers described can also be transfected into othermammalian cells such as HEK 293, BHK cells, murine NSO, and murine SP2/0cells.

Multimer Formation

We observed that these constructs, when transfected, cultured, andpurified may create proteins of the expected size in non-denatured anddenatured protein analysis. In addition, we observed that certaincompounds also exhibited larger bands which by size criteria aremultimers of the expected dimeric protein.

Formation of higher order compounds by selected stradomers was analyzedby SDS-PAGE followed by Western blot under non-reducing conditions (A)and reducing conditions (B). SDS-PAGE analysis shows formation of highmolecular weight compounds of stradomers G-002, G-010, and G-019 undernon-reducing conditions as compared to reducing conditions:

-   -   G-002: an approximately 35 KD band under reducing        condition—bands at approximately 70 KD (dimer) and 135 KD        (tetramer) under non-reducing conditions.    -   G-010: an approximately 45 KD band under reducing        condition—bands at approximately 90 KD (dimer) and 180 KD        (tetramer) under non-reducing conditions.    -   G-019: an approximately 35 KD band under reducing        conditions—bands at approximately 70 KD (dimer), 140 KD        (tetramer) under non-reducing conditions.

We anticipate that the tetrameric and other higher order multimers ofthe dimerized protein will contribute significantly to the biologicalactivity of the compound as measured by the immature Dendritic Cellassay (see below).

Stradomer Monomers, Stradomers, and Higher Order Multimers of StradomersMaintain Recognition Sites.

Each of the proteins in Table 3 are recognized by a rabbit anti-humanIgG (Fc) [Thermo Scientific 31789]. We conclude from this that each ofthese proteins maintains the recognition sites for this antibody.

Plasmon Resonance Imaging

The ability of the stradomers in Table 3 to bind FcγRIIIa was assessedusing surface plasmon resonance (SPR) technology (commercially availablethrough Biacore®). Human FcγRIIIa was directly immobilized via aminecoupling to a CM5 Biacore chip by diluting the ligand in Acetate pH5.0to a concentration of 5 ug/ml. Ligands were perfused over specified flowcell at a rate of 5 ul/min until an RU of 250 was reached. The flowcells were then blocked with ethanolamine. Stradomers and IVIG werediluted to 1000 nM in HBS-EP (0.01M HEPES pH 7.4; 0.15M NaCl; 3 mM EDTA;0.005% Surfactant P20) and serially diluted 500 nM, 250 nM, 125 nM andfinally 62.5 nM. A baseline sample containing only buffer (HBS-EP) wasalso included. A flow rate of 20 ul/min was used for all samples. Atotal of 60 uL of sample was injected, for an injection time of 3minutes. Regeneration was achieved by perfusing running buffer over flowcells for an extended period of time of approximately 10 minutes.

At 500 nM, the measured Req (equilibrium), relative to baseline for thestradomer G-010 construct was 24.9 RU when perfused over human FcγRIIIa,and the KD was 1.95c-7 using a 1:1 binding model. IVIG at 500 nM onhuman FcγRIIIa gave a Req of 63.6 RU and a KD of 1.89c-7 using a 1:1binding model. G-010 was therefore determined to bind to FcγRIIIa.Similar binding ability has been assessed on other biomimetic compounds.Here are some further examples:

1:1 w Mass Transfer Bivalent Fit Rmax Chi2 KD_((M)) KA_((1/M)) Rmax Chi2Controls: Neg. (mouse IgG2a) 2.05 0.451 4.7e−9 2.1e8 5.21 0.39 Pos.(IVIG) 87.7 6.8 1.9e−7 5.3e6 Biomimetics: 002 6.46 1.12 2.2e−8 4.9e716.7 0.82 004 30.2 1.74 4.8e−8 2.5e7 88.2 2.47 011 25.9 0.361 5.5e−61.8e7 57.4 0.15

We conclude that these proteins have varied ability to bind torecombinant FcγRIIIa by plasmon resonance analysis and that certaincompounds such as G-010 have a bivalent curve fit, consistent with thatseen by bivalent antibodies and indicating that the stradomer may havemulti-valent binding to FcγRIIIa.

Stradomers Mimic the Biological Effect of IVIG

The biological function of these stradomers was assessed. In order todetermine the ability of each of the stradomers in Table 3 to mimic thefunctional utility of IVIG in individuals with ITP, we developed an invitro assay using immature dendritic cells (iDC). The rationale forchoosing iDC as target cells was based on published data demonstratingthat adoptive transfer of DC from mice treated with IVIG, conferredprotection against the development of ITP to naïve animals. (Siragam, V.et al. Intravenous immunoglobulin ameliorates ITP via activatingFc[gamma] receptors on dendritic cells. Nat Med 12, 688 (2006)). In ourinitial studies, we evaluated the impact of coated, meaning fixed to theplate, recombinant Fc (rFc) and IVIG on the expression of a variety ofactivation, maturation and costimulatory markers on human CD14+ cells,cultured in the presence of IL-4 and GM-CSF. When compared to cellscultured in cytokines alone, cells exposed to coated IVIG or coated rFcdemonstrated striking enhancement of CD86 expression and down regulationof CD1a expression as well as a delay in CD11c upregulation.

Next, we determined whether the stradomers in Table 3 mimicked thedescribed effect of coated IVIG or coated Fc on iDC. These compoundswhen coated to the plate well walls and floors did mimic the effect:G-002, G-004, G-005, G-014, G-018, and G-019. These compounds whencoated to the plate well walls and floors did not mimic the effect:G-010, G-011, and G-012

These compounds when soluble did mimic the effect of coated IVIG orcoated Fc on iDC: G-002, G-010, G-014, G-018, and G-019. These compoundswhen soluble did not mimic the effect: G-004, G-005, G-011, and G-012.

Whether exposure of iDC to coated IVIG would influence subsequentresponses to pro-inflammatory stimuli can be tested.

We draw the following conclusions from these data:

-   -   i. that select stradomers, when coated on a tissue culture        plate, mimic the functional ability of coated IVIG to upregulate        CD86 and suppress CD1a expression on immature DC,    -   ii. that select stradomers administered at a low dose in a        soluble form mimic the functional ability of coated IVIG to up        regulate CD86 and suppress CD1a expression on iDC,    -   iii. that certain stradomers can induce phenotypic change in        both a soluble and coated form and that other stradomers, such        as G-010, can induce phenotypic change in a soluble but not a        coated form,    -   iv. that stradomers of differing structures can be biologically        active as evidenced by the Fc fragment stradomer formed from        G-002 and the cluster stradomer formed from G-010,    -   v. that structures larger than expected by dimerization of        stradomer monomers are seen on protein analysis and that these        multimeric structures may correspond with biological activity in        comparison to IVIG, and    -   vi. that stradomers formed from dimerized stradomer monomers can        demonstrate a bivalent fit on plasmon resonance consistent with        binding of multiple Fcγ receptors and suggesting the presence of        multimeric tertiary structures of the stradomers.

Example 3 Heat Aggregated Biomimetics are More Potent than IVIG

A stradomer is a biologically active mimetic of aggregatedimmunoglobulin and especially of the aggregated Fc fragments of thoseimmunoglobulin. In some instances, heat aggregation of the biomimeticsdescribed herein can increase biological activity. We conclude thatheat-aggregated biomimetics as herein described can be as potent asIVIG.

Example 4 Fc Fragment Exhibits Several Activities

The Fc fragment has been used as a positive control in experimentsdescribed above in which the protein is coated and thereby fixed toplastic thereby exhibiting biological behavior that mimics coated IVIG.The Fc fragment also can be used as a core stradomer unit such as whenit is attached to core moieties such as a liposome, a bead, or albumin.Further, we have demonstrated that the Fc fragment when cultured incertain expression systems and certain cell types, such as theInvitrogen FreestyleMax transient transfection system using CHO—S cells,can form higher order multimers on protein analysis, exhibit bivalentbinding pattern on plasmon resonance imaging, and exhibit profoundbiological activity in soluble form comparable to coated IVIG in theimmature DC assay. We conclude therefore that under certain carefullycontrolled conditions, the Fc fragment forms a Fc fragment stradomer.This effect may be due to post-translational modifications such asglycosylation changes.

Example 5 A Core Stradomer which is an Fc-Coated Bead May AlterPhagocytic Potential Relative to Uncoated Beads

PBMCs are isolated from the buffy coat of healthy donors usingFicoll-Hypaque density gradient centrifugation. After isolation, PBMCsare washed twice with PBS. CD14+ cells are then purified using MACSseparation column (Miltenyi). The purified cells are counted andresuspended to 2×10^5/ml RPM′ complete media containing 800 ug/ml GM-CSFand 5 ng/ml IL-4. The cells are then seeded in the wells of non-tissueculture but sterile 6-well plates. After seeding the CD14+ cells in thenon-tissue culture, polystyrene FITC microspheres (0.52 um) coated withor without saturating amounts of Fc or IVIG are added to the cells at a1:1 ratio and incubated for 6 days at 37° C., 5.0% CO2 and then analyzedfor phagocytosis of microspheres by FACS.

Both IVIG-coated beads and Fc-coated beads act as core stradomers andmay thereby alter phagocytotic potential relative to uncoated beads.

Example 6 Design of Immunologically Active Biomimetics with AlteredFcγRIII Binding Affinities

It has been shown that a shared set of residues of IgG1 are involved inbinding to all FcγRs. It has also been demonstrated that additionalresidues in IgG1 molecules are involved in the binding to both FcγRIIand FcγRIII. Some residues when altered inhibited binding of one or morereceptors. Interestingly, the specific double mutation of S298A/K334Aenhanced binding of FcγIIIa and reduced binding to FcγIIb. Thoseresidues have been noted on the stradomer construct shown in FIG. 16(using an asterisk at both amino acids). We therefore can use sitedirected mutagenesis to generate a stradomer molecule having thestructure encoded by SEQ ID NO: 17 but with the correspondingS298A/K334A mutations.

Example 7 Expression of Recombinant Proteins

Numerous expression systems exist that are suitable for use in producingthe compositions discussed above. Eukaryote-based systems in particularcan be employed to produce nucleic acid sequences, or their cognatepolypeptides, proteins and peptides. Many such systems are commerciallyand widely available.

In a preferred embodiment, the stradomers described herein are producedusing Chinese Hamster Ovary (CHO) cells which are well established forthe recombinant production of immunoglobulin proteins followingstandardized protocols. Alternatively, for example, transgenic animalsmay be utilized to produce the human stradomers described herein,generally by expression into the milk of the animal using wellestablished transgenic animal techniques. Lonberg N. Human antibodiesfrom transgenic animals. Nat Biotechnol. 2005 September; 23(9):1117-25;Kipriyanov S M, Le Gall F. Generation and production of engineeredantibodies. Mol Biotechnol. 2004 January; 26(1):39-60; See also Ko K,Koprowski H. Plant biopharming of monoclonal antibodies. Virus Res. 2005July; 111(1):93-100.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both incorporated herein byreference in their entirety, and which can be bought, for example, underthe name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ BACULOVIRUSEXPRESSION SYSTEM FROM CLONTECH®.

Other examples of expression systems include STRATAGENE®'s COMPLETECONTROL™ Inducible Mammalian Expression System, which utilizes asynthetic ecdysone-inducible receptor. Another example of an inducibleexpression system is available from INVITROGEN®, which carries theT-REX™ (tetracycline-regulated expression) System, an induciblemammalian expression system that uses the full-length CMV promoter.INVITROGEN® also provides a yeast expression system called the Pichiamethanolica Expression System, which is designed for high-levelproduction of recombinant proteins in the methylotrophic yeast Pichiamethanolica. One of skill in the art would know how to express vectorssuch as an expression construct described herein, to produce its encodednucleic acid sequence or its cognate polypeptide, protein, or peptide.See, generally, Recombinant Gene Expression Protocols By Rocky S. Tuan,Humana Press (1997), ISBN 0896033333; Advanced Technologies forBiopharmaceutical Processing By Roshni L. Dutton, Jeno M. Scharer,Blackwell Publishing (2007), ISBN 0813805171; Recombinant ProteinProduction With Prokaryotic and Eukaryotic Cells By Otto-Wilhelm Merten,Contributor European Federation of Biotechnology, Section on MicrobialPhysiology Staff, Springer (2001), ISBN 0792371372.

Example 8 Expression and Purification of Immunologically ActiveBiomimetics

Nucleic acid constructs described in Examples 1 and 2 are transfectedinto cell lines that do not naturally express Ig. The encodedpolypeptides are expressed as secreted proteins due to their secretoryleader sequences, which generally are removed by endogenous proteasesduring transport out of the cells or may be subsequently cleaved andremoved by techniques well known in the art. These secretedimmunologically active biomimetics are purified using Protein A or Histag chromatographic approaches well known in the art and size isverified by reducing and/or non-reducing SDS PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis).

Example 9 Expression and Purification of Immunologically ActiveBiomimetics for Large Scale Production

While various systems can be used to produce large amounts of a specificprotein including bacteria, insect cells or yeast, expression inmammalian cells can minimize problems due to altered glycosylation ofthe proteins. Mammalian cells like CHO cells have been used tooverproduce various proteins fused to an Ig backbone. The Fc domain inthe construct becomes a tag that permits subsequent purification fromthe cell supernatant using protein affinity column purification (Harris,C L, D M Lublin and B P Morgan Efficient generation of monoclonalantibodies for specific protein domains using recombinant immunoglobulinfusion proteins: pitfalls and solutions., J. Immunol. Methods268:245-258, 2002). Many fusion proteins are directly cloned in framewith the constant region of Ig, specifically the CH2 and CH3 partial Fcdomain monomers. A specific example of expression of interferon gammareceptor extracellular domain being expressed with Ig has been used toproduce large amounts of the protein with functional activity(Fountoulakis, M, C. Mesa, G. Schmid, R. Gentz, M. Manneberg, M. Zulauf,Z. Dembic and G. Garotta, Interferon gamma receptor extracellular domainexpressed as IgG fusion protein in Chinese hamster ovary cells:Purification, biochemical, characterization and stoichiometry ofbinding, J. Biol. Chem. 270:3958-3964, 1995).

Example 10 Design of Immunologically Active Biomimetics with Altered FcGlycosylation

By a method essentially the same as that described by Shields et al.with regard to homo-antibodies, de-fucosylated Fc domains can be made inmutant CHO cells lacking enzymatic activity for adding fucose to proteincarbohydrates. These are used to express stradomers with strongerFcγRIII binding affinities relative to a fucosylated form of the samemolecule. (Robert L. Shields, et al. Lack of Fucose on Human IgG1N-Linked Oligosaccharide Improves Binding to Human Fc Rill andAntibody-dependent Cellular Toxicity. J. Biol. Chem., July 2002; 277:26733-26740 (doi:10.1074/jbc.M202069200)).

It has been shown that changes in sialylation in the Fc N-glycan canincrease biological activity. Kaneko Y, Nimmerjahn F, Ravetch J V.Science. 2006 Aug. 4; 313(5787):627-8. Thus stradomer molecule havingaltered sialylation can be produced using similar methods.

Alternative means to altering glycosylation of stradomer Fc domainsinclude chemoenzymatic techniques for producing polypeptides with aspecific glycosylation structure. See, Li, B., Song, H., Hauser, S., andWang, L. X. 2006. A Highly Efficient Chemoenzymatic Approach TowardGlycoprotein Synthesis. Org. Lett. 8:3081-3084; See, also, InternationalPat. App. No. PCT/US07/70818.

Example 11 Fusion Constructs of FcγRIIIa (176 V/F) Polymorphism

As discussed previously, the anti-inflammatory activity of IVIG isdependent on primary interactions between the Fc domain and FcγRIIIa.These interactions can be effectively quantitated using (SPR) technologyto characterize both association and dissociation constants of theimmunologically active biomimetics with the two recognized polymorphicvariants of FcγRIIIa (176 V/F). In order to define the binding affinityand dissociation of our Fc domain monomeric control and stradomerconstructs, FcγRIIIa HIS tag fusion proteins will be produced in CHOcells with both V (SEQ ID NO:33) and F (SEQ ID NO:31) polymorphicvariants at position 176 (FIG. 20). These sequences can be put intopCDNA 3.1 and transfected into CHO cells. These FcγRIIIa fusion proteinsare purified from the supernatants from transfected cells using affinityNi²⁺ columns to purify the proteins. All FcγRIIIa fusion proteins arecharacterized by both cDNA sequencing and SDS PAGE.

Various other protocols in the art can be utilized to express FcγRIIIaand characterize interactions with immunologically active biomimetic.See, e.g., the materials and methods section of Robert L. Shields, etal. High Resolution Mapping of the Binding Site on Human IgG1 for FcγRI,FcγRII, FcγRIII, and FcRn and Design of IgG1 Variants with ImprovedBinding to the FcγR. J. Biol. Chem., February 2001; 276: 6591-6604(doi:10.1074/jbc.M009483200).

Example 12 Screening Immunologically Active Biomimetic Function In Vitro

To test the function of immunologically active biomimetics such as thosepresented in Example 1, an in vitro assay is designed to recapitulatethe mechanism by which it appears that native Fc domains reduceinflammation in vivo. It has recently been demonstrated that hIVIGinhibits the maturation of DCs and alters the secretion of IL-10, IL-12and TNF-alpha (Bayry, J, et al., Inhibition of maturation and functionof dendritic cells by intravenous immunoglobulin, Blood101(2):758-765(2003)). Our stradomers mediate effects on DCs similar tohIVIG. The inhibition of DC maturation and alterations in cytokinesecretion in vitro can serve as an effective means to define some of thebiological activities of many stradomer constructs. The stradomerconstructs described above may be further validated using the followingexperimental parameters:

TABLE 4 Experimental Outcome measure 1 Outcome measure 2 Group condition(FACS) ELISA/Elispot 1 None CD1a, 14, 40, 80, IL-10, IL-12, 83, 86,HLADR TNFa, IL-23 2 Soluble IVIG CD1a, 14, 40, 80, IL-10, IL-12, 83, 86,HLADR TNFa, IL-23 3 Fixed IVIG CD1a, 14, 40, 80, IL-10, IL-12, 83, 86,HLADR TNFa, IL-23 4 Soluble Fc CD1a, 14, 40, 80, IL-10, IL-12, 83, 86,HLADR TNFa, IL-23 5 Fixed Fc CD1a, 14, 40, 80, IL-10, IL-12, 83, 86,HLADR TNFa, IL-23 6 Soluble CD1a, 14, 40, 80, IL-10, IL-12, Stradomer83, 86, HLADR TNFa, IL-23 7 Fixed CD1a, 14, 40, 80, IL-10, IL-12,Stradomer 83, 86, HLADR TNFa, IL-23

In one preferred in vitro assay shown in Table 4, the impact on human DCphenotype of soluble, immunologically active biomimetics, havingappropriate binding affinities, is measured. Soluble non-cross-linkednatural sequence Fc domain constructs can serve as controls. Specific DCmarkers on the DC surface are evaluated including markers of activation(CD80, CD83 and CD86) as well as the FcγRs. See Prechtel A T, Turza N M,Theodoridis A A, Steinkasserer A. CD83 knockdown in monocyte-derived DCsby small interfering RNA leads to a diminished T cell stimulation. JImmunol. 2007 May 1; 178(9):5454-64. In addition, multiplex analysis canbe employed to evaluate the impact of our immunologically activebiomimetics on DC cytokine production. Jongbloed, Sarah L., et al.Enumeration and phenotypic analysis of distinct dendritic cell subsetsin psoriatic arthritis and rheumatoid arthritis. Arthritis Res Ther.2006; 8(1): R15 (Published online 2005 Dec. 16. doi: 10.1186/ar1864).Finally, to confirm DCs interact with monocytes as expected, control DCsand DCs exposed to immunologically active biomimetics are cultured withpurified monocytes and evaluated by flow cytometry for changes in thelevels of activating FcγRIIa receptors and other cell surfacedeterminants related to the activation state of the monocytes.

In particular embodiments, stradomers can decrease the FcγRIIa receptorspresent on an immune cell thereby increasing the ratio of inhibitoryFcγRIIb receptors to the FcγRIIa receptors which results in inhibitionof immune cell functions.

Example 13 Screening Immunologically Active Biomimetic Function In Vivo

Numerous autoimmune diseases such as idiopathic thrombocytopenicpurpura, multiple sclerosis, asthma, and inflammatory bowel diseaseshave established, art recognized animal models for in vivo testing. Wu GF, Laufer T M. The role of dendritic cells in multiple sclerosis. CurrNeurol Neurosci Rep. 2007 May; 7(3):245-52; Targan S R, Karp L C.Defects in mucosal immunity leading to ulcerative colitis. Immunol Rev.2005 August; 206:296-305. For example, multiple models of ITP arecurrently available. See, e.g., Crow A R, et al. IVIG inhibitsreticuloendothelial system function and ameliorates murine passiveimmune thrombocytopenia independent of anti-idiotype reactivity. Br JHaematol. 2001; 115:679-686. Immunologically active biomimetics designedto modulate the immune system, as appropriate for each specificautoimmune disease, can be validated in such in vivo models.Importantly, in many of these models, administration of hIVIG likelyresults in a foreign species (e.g. mouse) anti-human antibody responsewhich has the potential to obscure or create false positive productrelated anti-inflammatory effects.

We established a mouse model of Idiopathic Thrombocytopenic Purpuraaccording to the following methodology: Platelet counts were measured inC57BL6 mice by serial tail vein nicking. 10 ul. of blood was diluted in15 ul citrate buffer. Samples were then analyzed for absolute plateletcount on a HemaVet 950 cytometer. For mice in an ITP control group,starting day 2, every afternoon platelets were depleted by giving intraperitoneal injection of 2 ug Rat Anti-Mouse CD41 (MWReg30), ananti-platelet antibody from BD Biosciences pharmingen. Mice in the IVIGpretreatment control group received 2 g/kg (40 mg/mice) human IVIG byi.p. injection every morning and the same dose MWReg30 as the ITPcontrol group. We determined that IVIG is highly protective of plateletcount in this model of induced ITP and conclude that this model isuseful for testing stradomers against IVIG for relative degree ofprotection from platelet count decreases. A stradomer can be assessed inthis model at various concentrations to assess protection relative toIVIG as follows:

Groups in an Experiment

-   -   1) Control—No ITP, No IVIG    -   2) ITP control group—2 ug MWReg30 every evening starting day 2    -   3) IVIG pretreatment group—40 mg IVIG every morning and 2 ug        MWReg30 every evening starting day 2    -   4) Stradomer equivalent to 10^12 Fc domains IV every morning    -   5) Stradomer equivalent to 10^11 Fc domains IV every morning    -   6) Stradomer equivalent to 10^10 Fc domains IV every morning    -   7) Stradomer equivalent to 10^9 Fc domains IV every morning    -   8) Stradomer equivalent to 10^8 Fc domains IV every morning    -   9) Stradomer equivalent to 10^7 Fc domains IV every morning    -   10) Stradomer equivalent to 10^6 Fc domains IV every morning

Example 14 Validating Immunologically Active Biomimetic Efficacy In Vivofor Treating ITP

In another murine model of ITP, mice deficient in normal B cell functioncan be used. Deficiency in normal B cell function serves to eliminatethe idiotype-antiidiotype effects of murine anti-human Fc fragmentantibodies that would be generated by the administration of human Fcfragment or Fc partial fragment to a mouse and consequent false positiveresults. The deficiency in B cell function can be generated, forexample, through the administration of anti-B cell antibodies or occursin genetically engineered mice such as the m chain-knock out mouse(Jackson Labs strain B6.129S2-Igh-6^(tm1Cgn)/J) that are deficient inmature B-cells.

The immune system of immunodeficient mice is reconstituted with eitherunmodified or B cell depleted PBMCs from immunocompetent animals. Theseanimals are subsequently treated with anti-platelet antibodies to mimicITP using well defined techniques in the art. Animals are then treatedwith immunologically active biomimetics according to the followingscheme:

TABLE 5 In vivo efficacy of [hIgG₁ Fc domain - hIgG₁ Fc domain] (SEQ IDNO.: 22) immunologically active biomimetics for the treatment of ITP.PBMC's used to Animal reconstitute Outcome Group # mice TreatmentMeasure 1 5 None IgG1 Fc Platelet Count 2 5 None IgG1 Fc - IgG1 PlateletCount Fc Stradomer 3 5 Unmodified IgG1 Fc Platelet Count 4 5 UnmodifiedIgG1 Fc - IgG1 Platelet Count Fc Stradomer 5 5 B cell IgG1 Fc PlateletCount depleted 6 5 B cell IgG1 Fc - IgG1 Platelet Count depleted FcStradomer 7 5 Unmodified hIVIG Platelet Count

It is anticipated that groups 1 and 2 will not develop ITP upon antibodyinfusion as they do not have the B cells to produce anti-plateletantibodies necessary for platelet destruction. In groups 3 and 4, it isexpected that both the {hIgG₁ Fc-hIgG₁ Fc} stradomer polypeptide and thehIgG₁ Fc monomer polypeptide effectively ameliorate ITP becauseendogenous murine antibodies react with hIgG₁ Fc domain epitopes tocrosslink the hIgG₁ Fc monomer polypeptides. In contrast, in the absenceof endogenous murine antibodies, the {hIgG₁ Fc-hIgG₁ Fc} stradomerpolypeptide (group 6) is more effective than the uncross-linked IgG₁ Fcmonomer polypeptide (group 5) in ameliorating ITP. Group 7 serves as apositive control for treatment effect.

Example 15 Treating Patients with ITP Using Intravenous Formulations ofStradomer Proteins (SEQ ID NOs: 18 & 22)

Treatment protocols for ITP with the exemplary stradomer proteinsencoded by SEQ. ID. NO: 17 & 21 are utilized in a manner trackingstandard guidelines for ITP hIVIG therapy such as the ExecutiveCommittee of the American Society of Hematology practice guideline forthe diagnosis and management of primary immune thrombocytopenic purpura.See George, J N, et al. Idiopathic thrombocytopenic purpura: a practiceguideline developed by explicit methods for the American Society ofHematology. Blood. 1996 Jul. 1; 88(1):3-40; See also, the 2000guidelines by Italian pediatric hematologists, the 2003 Britishhematologists guidelines and the 2006 Japanese pediatric hematologistsguidelines. Alternatively, the stradomer IV protocols for ITP mayinclude an initial administration phase with dosages of about 0.1 toabout 0.001 times the above treatment protocol dosages. The initial lowdose phase is designed to minimize any short term pro-inflammatoryeffects of the stradomer administration while still being sufficient toinduce a long term anti-inflammatory effect, which is subsequentlyenhanced and maintained by the second phase standard dosing describedabove. The rationale for this alternative approach is that someembodiments of a stradomer may have both a short term inflammatoryeffect as well as a long term anti-inflammatory effect throughdecreasing the expression of FcγRIIa. An initial low dose (or initiallow doses) can be used to stimulate the long term anti-inflammatoryeffect while minimizing the short term inflammatory effect.

The effective stradomer dose is generally from about 0.01% to about 15%of the effective hIVIG dose, more preferably, about 0.1% to about 3% ofthe effective hIVIG dose. The effective hIVIG dose in ITP is generallyin the range of about 100 mg/Kg to about 2 grams/Kg administered every10-21 days.

The stradomer intravenous formulation will be substantially the same asFDA approved hIVIG formulations but may exclude the stabilizers presentin some hIVIG formulations. See, e.g., the product insert for GammagardS/D, distributed by Baxter Healthcare Corporation and approved for ITPtherapy by the FDA.

Example 16 Treating Patients with ITP Using IntraperitonealAdministration of a Core Stradomer

Treatment protocols for ITP with exemplary stradomer proteinsrepresenting Fc fragments fixed to a core moiety such as a liposome areutilized by intraperitoneal administration with dosages of about 1% toabout 0.001% of standard intravenous IVIG protocol dosages. Therationale for this alternative approach is that core stradomerscomprised of fixed Fc fragments delivered in a stable formulation to theintraperitoneal cavity will make available the multiple Fc domains toaffect monocyte-derived effector cells similarly to IVIG but atsubstantially lower doses.

Example 17 Design of Immunologically Active Biomimetics (Stradobodies)

Two stradobodies have been constructed and transfected. For eachstradobody, encoding cDNA was synthesized from total RNA made fromhybridoma cell lines expressing the antibody of interest. Establishinghybridoma cell lines is well known in the art. Amplification of cDNA ofinterest encoding the antibody heavy and light variable regions was doneby BD SMART™ RACE amplification kit (Clontech CA). Numerous othermethods are available to generate cDNA encoding the heavy and lightchains for variable regions of antibodies (Sassano, M. et. al., 1994.Nucleic Acids Res. May 11; 22(9):1768-9; Jones, S. T., Bendig, M. M.,1991. Biotechnology (NY) January: 9(1):88-9.) To generate thestradobodies the heavy chain variable regions are fused to the stradomerconstructs by either sewing by overlap extension with PCR (Hutton andPease) or utilize existing compatible restriction sites to fuse theappropriate fragments. Stradobody proteins are expressed in CHO—S cellsand isolated from cell supernatants by protein A column affinitypurification. Binding of the purified stradobodies to the antigen ofinterest is confirmed by flow cytometry binding studies utilizing celllines expressing the antigen.

A standard ADCC assay employing NK cells as effectors and antigenexpressing tumor cells as targets at various effector-to-target ratiosis employed to compare the potential of the stradobody and themonoclonal antibody (Mab) that shares the same Fab region to induce ADCCagainst high and low antigen expressing tumor cell lines. Stradobodiesare selected for development that demonstrate similar results to thepaired Mab in the NK assay against the high epitope expressing cell linebut superior results to the paired Mab in the NK assay against the lowepitope expressing cell line.

Example 18 Treating Patients with Breast Cancer Using IntravenousFormulations of Stradobody Containing the Antigen-Binding Domain ofTrastuzumab

Treatment protocols for breast cancer with the exemplary stradobodycontaining a Fab that is or is similar to the Fab from the marketedproduct trastuzumab having activity against the her2/neu epitope areutilized in a manner tracking standard guidelines for breast cancertherapy. See Romond, E H et. al. Trastuzumab plus Adjuvant Chemotherapyfor Operable HER2-Positive Breast Cancer. NEJM. 2005 Oct. 20;353:1673-1684; Seidman, A D et. al. Weekly Trastuzumab and PaclitaxelTherapy for Metastatic Breast Cancer With Analysis of Efficacy by HER2Immunophenotype and Gene Amplification. Journal of Clinical Oncology.Vol 19, Issue 10 (May), 2001: 2587-2595; Vogel, C L et. al. Journal ofClinical Oncology. Vol 20, Issue 3 (February), 2002:719-726

It is anticipated that the effective stradobody dose will generallyrange from about 1% to about 500% of the effective monoclonal antibodywhose Fab is the same as the stradobody, more preferably, about 50% toabout 100% of the effective monoclonal antibody dose. The effectivemonoclonal antibody dose in clinical cancer treatment varies. For theHer-2 neu monoclonal antibody the dose is generally in the range ofabout 2 mg/Kg to about 4 mg/Kg administered every 7-21 days.

Example 19 Treating Patients with Head and Neck or Colon Cancer UsingIntravenous Formulations of Stradobody Containing the Antigen-BindingDomain of Cetuximab

It is anticipated that treatment protocols for breast cancer with theexemplary stradobody containing a Fab that is or is similar to the Fabfrom the marketed product cetuximab having activity against the EGFRepitope can be utilized in a manner tracking standard guidelines forhead and neck and colon cancer therapies. See Robert, F et. al. Phase IStudy of Anti-Epidermal Growth Factor Receptor Antibody Cetuximab inCombination With Radiation Therapy in Patients With Advanced Head andNeck Cancer. Journal of Clinical Oncology, Vol 19, Issue 13 (July),2001: 3234-3243; Bonner, J A et. al. Cetuximab prolongs survival inpatients with locoregionally advanced squamous cell carcinoma of headand neck: A phase III study of high dose radiation therapy with orwithout cetuximab. Journal of Clinical Oncology, 2004 ASCO AnnualMeeting Proceedings (Post-Meeting Edition). Vol 22, No 14S (July 15Supplement), 2004: 5507; Shin, D M et. al. Epidermal Growth FactorReceptor-targeted Therapy with C225 and Cisplatin in Patients with Headand Neck Cancer. Clinical Cancer Research Vol. 7, 1204-1213, May 2001;Cunningham, D et al. Cetuximab Monotherapy and Cetuximab plus Irinotecanin Irinotecan-Refractory Metastatic Colorectal Cancer. NEJM. Volume351:337-345, 2004.

It is anticipated that the effective EGFR/HER1 stradobody dose willgenerally range from about 1% to about 500% of the effective monoclonalantibody whose Fab is the same as the stradobody, more preferably, about50% to about 100% of the effective monoclonal antibody dose. Theeffective monoclonal antibody dose in clinical cancer treatment varies.For the EGFR monoclonal antibody the dose is generally in the range ofabout 250-400 mg/square meter which is about 5 mg/Kg-25 mg/Kgadministered every 7-21 days.

Example 20 Increased Multimerization by Altered Glycosylation MayIncrease Immunologically Active Biomimetic Activity

The glycosylation patterns of expressed proteins are dependent on thecell line in which the protein is expressed. The Chinese Hamster Ovariancell (CHO cell) commonly used for protein expression and purificationresults in a glycosylation pattern that is different from, for example,the HEK 293 cells which are of human origin and also is commonly usedfor protein expression of endogeneous proteins. As the bindingproperties of Fc fragments and cluster stradomer units can be affectedby the glycosylation pattern, increased multimerization and thereforeincreased biological activity of the expressed peptides can be achievedby expression in cell lines other than CHO or in cell lines includingCHO that are genetically altered to change the glycosylation pattern toan N-glycan that promotes increased aggregation between Fc fragments orFc domain-containing peptides. Increased multimerization of Fc fragmentor selected cluster stradomer units by altering glycosylation patternsmay increase the ability of immunologically active biomimetics to mimicthe effects of hIVIG.

Example 21 Does Exposure of Mature DC (mDC) to IVIG or rFcF (RecombinantFc Fragments) Alter their Phenotype?

The rFCF fragments from human IgG1 to be used in this experiment wereproduced by standard recombinant protein technology. The two chains ofthe human rFCF each consisted of the hinge region (15 amino acids), theCH2 domain (110 amino acids), and the CH3 domain (106 amino acids) ofhuman IgG1 heavy chain.

CD14+ cells can be isolated from peripheral blood mononuclear cells(PBMC) obtained from the blood of a healthy human donor using a MiltenyiMACS separation column. The cells are cultured at a final concentrationof 2×10⁵/mL in GM-CSF (800 IU/mL) and IL-4 (5 ng/mL) for 5 days at 37°C. The media in all cultures is refreshed on day 3 of culture. At day 5,lipopolysaccharide (LPS; 10 μg/ml) is added to appropriate cultures toinduce maturation to a mature DC. Mature DCs are known in the art not toexpress substantive levels of the CD16, CD32 or CD64. The cells are thencultured for an additional two days and aliquots are analyzed for CD11c,CD80, CD83, CD86, CD1a, and CD14 expression by two dimensionalfluorescence flow cytometry (FFC). The remaining cells cultured with LPSare then placed in wells with soluble or coated IVIG or human rFcF (allat 10 μg/mL) for 24 hours at 37° C., harvested and analyzed forexpression of the markers listed above by two-dimensional FFC.

Experimental groups are as follows:

-   -   (1) CD14+ cells; GM-CSF; IL-4; no LPS (“7d-LPS”)    -   (2) CD14+ cells: GM-CSF; IL-4; LPS (“7d+LPS”)    -   (3) CD14+ cells; GM-CSF; IL-4; LPS; coated IVIG (“cIVIG”)    -   (4) CD14+ cells; GM-CSF; IL-4; LPS; soluble IVIG (“sIVIG”)    -   (5) CD14+ cells; GM-CSF; IL-4; LPS; coated rFcF (“cFc”)    -   (6) CD14+ cells; GM-CSF; IL-4; LPS; soluble rFcF (“sFc”)    -   (7) CD14+ cells; GM-CSF; IL-4; LPS (“Control”)

Example 22 Does Exposure of iDC to Coated IVIG Inhibit Phagocytosis ofOpsonized Red Blood Cells

CD14+ cells are purified from human PBMC of a healthy human donor asdescribed in Example 21 and cultured at 37° C. for 6 days with GM-CSFand IL-4 at the concentrations indicated in the previous examples and inthe presence or absence of coated or soluble IVIG. The cells areharvested and then incubated at either 37° C. or 4° C. for two hourswith Rho-positive human red blood cells that are uncoated or coated withfluorescein isothiocyanate (FITC) conjugated anti-D antibody. Afterincubation with red blood cells, CD14+ cells are stained forAPC-conjugated CD1a. Phagocytosis is then evaluated by two dimensionalFFC measuring side light scatter (SSC-A), forward light scatter (FSC-A),FITC fluorescence (FITC-A), and APC fluorescence (CD1a).

Example 23 Does Exposure to Coated IVIG Decrease the Ability of iDC toStimulate an Allogeneic Mixed Lymphocyte Reaction

CD14+ cells are isolated from the blood of a healthy human donor asdescribed in the previous examples. They are then cultured at 37° C. for6 days with GM-CSF and IL-4 in the presence or absence of soluble andcoated IVIG. The concentrations of all these reagents are as describedin above. The cells are then harvested and plated into the wells of 96well microtiter tissue culture plates at various numbers (with thehighest dose being 2.5×10⁴ per well). CD3+ T cells are purified from thePBMC of a second human donor that was HLA incompatible with the donorfrom which the CD14+ cells are isolated. The T cells are added to eachof the wells of the 96 well tissue culture plates (10⁵ T cells perwell). After five days of co-culture, 1 μCi of ³H-thymidine is added toeach of the culture wells. The cultures are then incubated for a further6 hours and incorporation of the ³H-thymidine (“cpm”) is measured as anindication of the degree of cell proliferation in the cultures. Threedifferent iDC stimulator populations are tested: one generated byculture with GM-CSF and IL-4 only, one generated by culture with GM-CSF,IL-4, and coated IVIG, and one generated by culture with GM-CSF, IL-4,and soluble IVIG.

Example 24 Effect of Exposure of iDC to Coated and Soluble rFcF and IVIGon Cytokine Expression by the iDC and mDC

Cultures containing CD14+ cells, GM-CSF, and IL-4 and either rFcF(coated or soluble) or IVIG (coated or soluble) are set up under theconditions described in the previous examples. Instead of testing thecells for expression of cell surface markers, phagocytic ability, or theability to stimulate allogeneic MLRs, the cytokines the cells produceare measured. It is expected that coated rFcF will modulate cytokineproduction by the cells in a manner similar to IVIG but not similar tosoluble rFcF. Thus, it is expected that the level of cytokines thatinhibit inflammatory responses (e.g., interleukin-4, interleukin-6, andinterleukin-12) will be enhanced by exposure of the cells to coatedrFcF. Moreover, it is expected that exposure of the cells to coated rFcFwill result in a decrease in the level of production by the cells ofcytokines that enhance inflammatory responses (e.g., interferon,interleukin-23, and tumor necrosis factor-I).

Example 25 Recombinant Mouse Fc Fragments

Recombinant Fc fragments (rFcF) from mouse IgG2a were produced usingstandard cloning and recombinant protein expression techniques. The twochains of the mouse rFcF each consisted of the hinge region (21 aminoacids), the CH2 domain (110 amino acids), and the CH3 domain (107 aminoacids) of mouse IgG2a heavy chain. The mouse IgG2a was active in thehuman iDC assay when coated to the walls and floors of plate wells.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps. All U.S. and foreign patents, patentapplication publications, and non-patent literature (including, but notlimited to, abstracts, scientific journal articles, books, productliterature and manuals) referred to or cited herein are herebyincorporated by reference in their entireties.

LIST OF REFERENCES

The following references are incorporated by reference in theirentirety.

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What is claimed is:
 1. A cluster stradomer comprising two or moremultimerized cluster stradomer units, wherein each of said clusterstradomer units comprises from amino terminus to carboxy terminus, (a)at least one multimerization region which multimerizes the clusterstradomer units to form the cluster stradomer wherein the multimerizingregion is an IgG2 hinge and (b) at least one Fc domain comprising ahinge, CH2 and CH3 domain from IgG1 wherein the cluster stradomer iscapable of specifically binding to at least two Fc receptors.
 2. Thecluster stradomer of claim 1 wherein said cluster stradomer does notcontain an antigen-binding Fab domain.
 3. The cluster stradomer of claim1, comprising two multimerized cluster stradomer units.
 4. The clusterstradomer of claim 1, comprising three multimerized cluster stradomerunits.
 5. The cluster stradomer of claim 1, comprising four multimerizedcluster stradomer units.
 6. The cluster stradomer of claim 1, comprisingfive or more multimerized cluster stradomer units.
 7. The clusterstradomer of claim 1 wherein each of the cluster stradomer units iscapable of specifically binding to at least one FcR.
 8. The clusterstradomer of claim 1 wherein the at least one FcR is human Fcγ receptorI, human Fcγ receptor II, human Fcγ receptor III, or FcRN.
 9. Thecluster stradomer of claim 8 wherein the human Fcγ receptor III is humanFcγRIIIa.
 10. The cluster stradomer of claim 1 comprising clusterstradomer units each of which comprise a homodimer of the protein of SEQID NO:
 61. 11. A homodimeric cluster stradomer unit wherein each monomerof the homodimer comprises from amino terminus to carboxy terminus: (a)a multimerizing domain monomer comprising an IgG2 hinge that whenhomodimerized is capable of multimerizing the cluster stradomer unit;and (b) an Fc domain monomer comprising an IgG1 hinge monomer, an IgG1CH2 monomer, and a IgG1 CH3 monomer wherein the homodimer is capable ofspecifically binding to at least one Fc receptor and multimerizes toform a compound capable of binding to two or more Fc receptors.
 12. Thehomodimeric cluster stradomer unit of claim 11 wherein each monomer ofthe homodimer comprises the protein of SEQ ID NO: 61.